Posttranscriptional regulation of uridine diphosphate glucuronosyltransferases.

Review

Expert Opin. Drug Metab. Toxicol. Downloaded from informahealthcare.com by University of Otago on 07/05/15 For personal use only.

Posttranscriptional regulation of uridine diphosphate glucuronosyltransferases 1.

General introduction

2.

Gene structure and function of the UGTs

3.

UGT phylogeny and evolution

4.

UGT tissue-specific expression

5.

UGT translation

6.

Latency

7.

Posttranslational modifications affecting UGT activity

8.

Allosteric interactions

9.

Intra- and inter-family protein--protein interactions

10.

Ontogenetic regulation of UGT

11.

Conclusion

12.

Expert opinion

Zoe Riches & Abby C Collier† University of British Columbia, Faculty of Pharmaceutical Sciences, Vancouver, BC, Canada

Introduction: The uridine diphosphate (UDP)-glucuronosyltransferase (UGT) superfamily of enzymes (EC 2.4.1.17) conjugates glucuronic acid to an aglycone substrate to make them more polar and readily excreted. In general, this reaction terminates the activities of chemicals, drugs and toxins, although occasionally a more active or toxic species is produced. Areas covered: In addition to their well-known transcriptional responsiveness, UGTs are also regulated by posttranscriptional mechanisms. Here, the authors review these mechanisms, including latency, modulation of co-substrate accessibility and binding, dimerization and oligomerization, protein--protein interactions, allosteric inhibition and activation, posttranslational structural and functional modifications and developmental switching for UGTs. Expert opinion: Posttranscriptional regulation of UGTs has traditionally received less attention than nuclear regulation, in part because mechanisms involving ribosomes and endoplasmic reticula are challenging to investigate. Most promising of the posttranscriptional mechanisms reviewed are likely to be effects on co-substrate (UDP-glucuronic acid) transport and availability and structure--function changes to UGT proteins through, for example, glycosylation and phosphorylation. Although classical biochemistry continues to illuminate many aspects of UGT function, advances in proteomics and structural biology are beginning to assist in the determination of posttranscriptional regulation mechanisms for UGTs. Keywords: conjugation, drug metabolism, enzyme activity, glucuronidation, glycosylation, phosphorylation Expert Opin. Drug Metab. Toxicol. (2015) 11(6):949-965

1.

General introduction

The uridine diphosphate glucuronosyltransferase super family The uridine diphosphate (UDP)-glucuronosyltransferase (UGT) superfamily of enzymes (EC 2.4.1.17) performs the metabolic reaction known as glucuronidation. Glucuronidation is a SN2 addition reaction, where the UGT using the co-substrate UDP-glucuronic acid (UDPGA), catalyzes the conjugation of a glycosyl sugar to an aglycone substrate in a b-D configuration [1-4]. Figure 1 shows the topology of the enzyme with transporters in the endoplasmic reticulum (ER) membrane. Although commonly called a ‘cofactor,’ UDPGA is more correctly a co-substrate as UGTs hydrolyze UDPGA into UDP and glucuronic acid. Addition of the glucuronide side chain makes the substrate larger, more polar and more water soluble. The typical consequence of this structural modification is that the substrate becomes inactive and is excreted from the system [3]. Another less common consequence is the creation of a biologically active, toxic metabolite -- commonly through the production of acyl glucuronides that internally rearrange [5]. UGT metabolism is integral in vertebrate detoxification and homeostasis. Multiple UGT enzymes allow a cell to respond to diverse physiological and 1.1

10.1517/17425255.2015.1028355 © 2015 Informa UK, Ltd. ISSN 1742-5255, e-ISSN 1744-7607 All rights reserved: reproduction in whole or in part not permitted

949

Z. Riches & A. C. Collier

2.

Article highlights. .

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The uridine diphosphate (UDP)-glucuronosyltransferases (UGTs) are, arguably, the most important superfamily of endo- and xenobiotic-metabolizing enzymes. The UGTs are genetically, transcriptionally and posttranscriptionally regulated. Recently a study of posttranscriptional regulation has revealed glucuronidation is controlled, at least in part, at the protein level. Mechanisms of posttranscriptional regulation include: transport and affinities of the co-substrate UDP-glucuronic acid (and other glycosyl sugars), allosteric enzyme inhibition and activation, protein dimerization and oligomerization, protein--protein interactions, and structure-function changes by, for example, glycosylation and phosphorylation. Posttranscriptional regulation of UGTs can be genetically or environmentally driven and may be as important as transcriptional controls.

This box summarizes key points contained in the article.

pathophysiological stimuli. A broad and overlapping substrate specificity characterizes the UGTs and these enzymes are present in mammals, fish, birds and insects [3]. The UGTs are responsible for the biotransformation of many endogenous compounds, including bilirubin, steroid hormones, bile acid, retinoids and thyroid hormones [3]. The UGTs also play a remarkable role in homeostasis of endogenous steroids, including various progestogen, androgen and estrogen hormones and their metabolites, albeit with lower substrate capacity than the sulfotransferases [3]. Glucuronidation is the most widespread conjugation reaction in humans probably due in part to the abundant amounts of the cofactor UDPGA and the ubiquitous expression of the UGTs [6,7]. Moreover, one estimate places up to 35% of all Phase II conjugation in the human body as being performed by UGTs [8]. Common pharmaceutical substrates of UGTs include acetaminophen, analgesics (morphine and codeine), anti-viral drugs (3¢azido-3¢-deoxythymidine [AZT]), anti-psychotics and anti-depressants (lorazepam, trifluoperazine, imipramine), nonsteroidal anti-inflammatory drugs (ibuprofen and naproxen), and chemotherapy drugs (irinotecan) [3,6,7,9,10]. Excellent reviews of UGT transcriptional regulation have recently been published and the ability of UGT enzymes to be regulated by endogenous and exogenous transcription factors is well-established [11-13]. Moreover, the potential for epigenetic regulation of UGTs at the level of methylation and histone modification is emerging from our laboratory for UGT2B15 (unpublished data) and others’ for UGT1A1 [14,15] and UGT1A10 [16]. In addition to their well-known transcriptional responsiveness, UGTs are also modified by posttranscriptional mechanisms. Despite this, the literature lacks a recent, comprehensive exposition of posttranscriptional regulation of UGTs. 950

Gene structure and function of the UGTs

The UGT superfamily nomenclature is organized based upon divergent evolution, with each gene given the symbol UGT, an Arabic number denoting the family, a letter denoting the subfamily, and an Arabic number for each gene within that subfamily (Figure 2). The UGT superfamily consists of five subfamilies in humans: UGT1A, 2A, 2B, 3A and 8A [17-19]. Nomenclature for rodents is the same, except that the genes are given lowercase letters (i.e., Ugt) as per the convention in the Mouse Genome Informatics database. The UGT genes are, in general; nonhomologous between the many species that express UGTs (e.g., rodents, nonhuman primates, rabbits, fish, etc.). Spanning ~ 200 kilobases (kb), the human UGT1 locus consists of a single gene located on chromosome 2q37.1 [2,10]. This gene is composed of a region of 13 individual first exon/promoter pairs (1A1, 1A2p, 1A3, 1A4, 1A5, 1A6, 1A7, 1A9, 1A13p, 1A10, 1A8, 1A11p and 1A12p), upstream to a region of common exons (2, 3, 4, 5a and 5b) with a shared promoter. One first exon, encoding 288 amino-terminal amino acids, is alternatively spliced to the grouped common exons, encoding 246 carboxyl terminal amino acids, to generating one of the 13 possible mRNA isoforms, each with unique 5¢ sequences, but identical 3¢ ends [20]. Of these 13 possible mRNA isoforms it is predicted that 9 translate into functional protein (UGT1A1, 1A3, 1A4, 1A5, 1A6, 1A7, 1A9, 1A10 and 1A8). This mechanism of UGT diversity, combining first and common exons, is also conserved in mouse and rat UGTs [21]. The first exons of UGT1A encode the substrate-binding domains. Alternative exon assembly allows for a cell to generate UGTs in response to different stimuli. Conversely, the grouped UGT1A common exons encode the conserved UGT functional domain, including the UGT cofactor, UDPGA, binding site as well as the UGT membraneanchoring region, 245 C-terminal amino acids. This coding region is essential for the basic glucuronic acid transferase function of UGTs. Furthermore, the UGT signature sequence, a specific order of nucleotides characteristic of UGTs, is also found in the shared common exons (amino acids 289 -- 533) [10,20,22]. The UGT1A isoforms are expressed ubiquitously throughout the body in a tissue-specific manner with, for example UGT1A7, 1A8 and 1A10 being only found in the gastrointestinal (GI) tract [23,24]. The UGT2 locus is present on chromosome 4q13 and encodes members of the UGT2A and 2B subfamilies. Similar to the UGT1A family, UGTs 2A1 and 2A2 are encoded by unique first exons joined to common exons 2 -- 6 [2]. In contrast, UGT2A3 has six unique exons and does not share common exons with any other UGT isoform [2]. Originally believed to exist only in the nasal mucosa, recent data demonstrate that UGT2A isoforms are also expressed and active in the lung [25,26]. In contrast, the human UGT2B subfamily consists of multiple unique genes each with six exons that

Expert Opin. Drug Metab. Toxicol. (2015) 11(6)

Posttranscriptional UGT regulation

Glucuronide conjugate

Lumen

UDP-Glucuronosyltransferase


UDP-GlcUA uptake transporter

Glucuronide efflux transporter

Substrate

UDP-GlcUA

Cytosol

Figure 1. Hypothetical topology model for human UGT, with the active site localized on the luminal aspect of the ER. A UDPGlcUA transporter facilitates the transfer of cofactor across the membrane. Figure reproduced with permission from [19]. ER: Endoplasmic reticulum; UDP: Uridine diphosphate; UDPGlcUA: Uridine diphosphate glucuronic acid; UGT: Uridine diphosphate glucuronosyltransferase.

independently encode for an individual UGT2B isoform. In total, there are seven human UGT2B isoforms (UGT2B4, 2B7, 2B10, 2B11, 2B15, 2B17 and UGT2B28) with five pseudogenes (UGT2B24p, 2B25p, 2B26p1, 2B26p2 and 2B27p) with both UGT2A and UGT2B distributed together over a 1500-kb region. Similar to the UGT1A gene structure, the first UGT2B exon encodes the substrate-binding domain, while the remaining exons encode the UDPGA-binding and transmembrane domains [10]. The UGT3 locus is found at chromosome 5p13.2 with UGT3A1 and 3A2 being the members expressed in this subfamily. Consisting of seven exons each and separated by ~ 76 kb, the UGT 3A isoforms have only recently been described via sequencing of human and rodent genomes [27-29]. They are only moderately homologous to other human UGTs (~ 30%) and endogenous substrates for UGT3A isoforms identified to date are bile acids, while xenobiotic substrates include planar phenols such a 4-nitrophenol and 4-methylumbelliferone [27-29]. Unlike the UGT1 and UGT2 isoforms, the UGT3A isoforms not only perform glucuronidation, but also possess galactosidase, glucosidase and xylosidase activities and can utilize UDP-galactose, UDP-N-acetyl-glucosamine and UDP-xylose as cofactors/co-substrates. The UGT8A family is orphan in terms of xenobiotics. Sequence analysis indicates that there is a single gene in humans encoding UDP galactose ceramide galactosyltransferase, consisting of five exons on chromosome 4q26 [30]. The enzyme UGT8A has a critical role in the biosynthesis of the

glycosphingolipids, cerebrosides and sulfatides. Although its role in drug metabolism is relatively undefined, the most recent data indicate that UGT8 enzymes can metabolize bile acids by galactosidation [31]. 3.

UGT phylogeny and evolution

Ancestrally, it is believed that UGTs developed in vertebrates to detoxify ingested environmental chemicals, mainly plant polyphenolic phytoalexins, antimicrobial compounds and plant phytoestrogens [32]. These phytotoxins are antimicrobial compounds generated in plant cells. As a result of a plant rich diet, it is believed that UGTs evolved to clear the body of various plant compounds, and this eventually came to include endogenous compounds. The multiple UGT families arose > 400 million years ago [32]. Evolutionarily, the current theory described adaptation by changing UGT substratebinding capabilities, through genetic polymorphisms and chromosomal reassortment, in response to environmental stimuli. Today, nonsynonymous mutations have modified UGTs to glucuronidate an ever-changing repertoire of substrates, including natural and man-made xenobiotics [21,32,33]. A characteristic of UGTs is that individual enzymes display not only some unique substrate specificities/selectivities, but also a large, promiscuous and overlapping substrate repertoire. Duplication of exons in the UGT1A family and segmental chromosome duplication in the UGT2B family leads to the


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UGT1A1 71% UGT1A3

93%

UGT1A5

94%

UGT1A2p 67%

UGT1A4 UGT1A6

66%

1

93% 89%


83%

UGT1A7 UGT1A8 UGT1A10 UGT1A9 UGT1A13p UGT1A11p UGT1A12p

41%

UGT2B7 87%

94% 89%

85%

UGT2B11 UGT2B28 UGT2B10

B 2

78%

94%

UGT2B15 UGT2B17

59%

UGT2B4 UGT2A1 A UGT2A2

Figure 2. Phylogenetic tree for UGT isoenzymes. The dendrogram shows both UGT families that share < 50% of homology. Percentage values represent the homology between two groups or single isoenzymes at the amino acid level. Pseudogenes were not analyzed for homology. Figure reproduced with permission from [135]. UGT: Uridine diphosphate glucuronosyltransferase.

UGT’s adaptability toward many common environmental and endogenous compounds. Thirteen UGT1A first exons originated from gene duplication of ancestral exons UGT1A1 and UGT1A6 [22]. Human UGT sequence homology, corroborated by interspecies comparisons, indicates that there are two highly identical clusters of exons [20]. The first cluster, consisting of gene duplications of UGT1A1, includes UGT1A2p, 1A3, 1A4 and 1A5, which are 87 -- 92% homologous. These isoforms are the bilirubinlike metabolizing enzymes. Conversely, the second cluster, consisting of gene duplications of UGT1A6, includes UGT1A7, 1A8, 1A9, 1A11p, 1A12p and 1A13p, which are 67 -- 91% identical. These isoforms are responsible for metabolizing the phenol-like compounds [10,22]. Similarity of UGT1A first exons results in UGT1A isoforms sometimes having broad, overlapping and/or divergent substrate specificities. Furthermore, the high level of identity in UGT1A1 duplications indicates that 952

these duplications occurred more recently. It has been postulated that the UGT1A1 cluster, due to general inactivity and presence of pseudogenes, represents a set of enzymes that are still evolving. UGT evolution through gene duplication has led to a great diversity of UGT1A isoforms created by the sharing of alternative first exon promoters to an extent not seen in any other enzyme system [20]. The multiple UGT2B genes originated from segmental chromosome duplication [10]. This is indicated by the high sequence identity (> 70%) of each UGT2B gene as well as the conservation of complete coding regions. The UGT2B family is also associated with copy number variants, which can lead to gene duplication or deletion. Indeed UGT2B17 has been shown to be the most commonly deleted gene in the human genome and in turn is associated with a significant variation in expression levels between ethnic groups [34].




4.

UGT tissue-specific expression

Alternative use of duplicated first exons is the basis of diversity in UGT1A function and expression. Tissue-specific expression is orchestrated by interactions between transcription factors and the transcription initiation complex. While UGTs are expressed holistically, their detoxification and clearance role is primarily situated in liver hepatocytes, with secondary major sites being in the kidney and GI tract and other sites of UGT expression having relatively minor systemic roles, but potentially critical localized roles. A study of UGT mRNA expression in the liver shows that UGT2B4 and 1A1 have the most abundant mRNA expressed in the liver with UGT2B4 > UGT1A1 mRNA expression [35]. This is followed by UGT1A6 > 1A4 » 1A9 » 2B7 > 1A3. A hallmark of UGT expression is a wide range of individual variability in adults from 2.5 fold for UGT1A4 to 8- and 7-fold, respectively, for UGT1A1 and 1A6 mRNA [35]. Early studies demonstrated that UGT proteins in the human liver mature functionally in the first 2 years of life, but there are several-fold differences in protein levels, even for the same UGT isoenzyme [36]. Subsequently, in the developing human liver it has been demonstrated that the expression of UGT proteins in human liver is also very variable with UGT1A1, 1A4, 1A6 levels ranging four-, eight- and ninefold, respectively, for these isoforms in the adult liver [37-39]. Most recently, UGT isoenzymes have been quantified by mass spectrometry in human liver with the following relative abundance 1A1, > 2B4, > 2B15, > 1A4, > 2B10, > 1A9, > 2B17, > 1A6 and > 1A3, with variable expression of UGT2B17 such that it was not expressed in 5/16 livers tested [40]. However, it is very difficult to determine the relevance of absolute abundance studies, particularly in the face of recent data showing that liver disease (nonalcoholic fatty liver disease) can alter UGT protein mRNA, protein and activities [41]. In the intestine, several-fold differences in UGT isoforms have been reported. Notably, the proteins and their activity toward selective substrates varies between the jejunum, ileum and duodenum in adults and, in the case of UGT2B4 and UGT2B7 was up to sevenfold different between the three compartments [42]. Moreover, in human intestinal microsomes absolute quantification using mass spectrometry has revealed that levels of isoform proteins are UGT2B17 >> 1A1, 1A10, 2B7, 1A3 and 1A4 [40]. Extrahepatic metabolism, especially in the intestines, may lead to low bioavailability of certain drugs. This is not usually relevant for approved drugs because low bioavailability is compensated for in the dosing and formulation areas of drug development. However, in the context of GI-mediated glucuronidation dietary and environmental exposures may modulate UGT expression and activities and hence present a changing interface for drug disposition [43,44].

As mentioned, apart from the liver, glucuronidation also occurs in the adipose, bile ducts, brain, colon, gastrointestinal tract, kidneys, lungs, placenta, reproductive organs, testis, skin and stomach [10] with three UGT1A isoforms are expressed exclusively in the GI tract: UGT1A7, 1A8 and 1A10 [3]. In terms of UGT development in extrahepatic tissues, adults express higher levels of UGT2B7, 2B15 and 2B17 mRNA and protein than fetuses in kidneys and lungs, with UGT2B7 being the predominant [45] dominant isoform [45]. In the adult human kidney, levels of UGT proteins measured by mass spectrometry were UGT1A9 >> UGT2B7 >> UGT1A6 [40]. Of further interest, in androgen and estrogen responsive tissues, including adipose, breast, prostate, testis where they play important roles in the homeostasis of steroid hormones and may have implications in preventing the development of certain cancers in humans [46-49]. 5.

UGT translation

While the UGTs have become a subject of intense research, comparatively little is known about their structure. The intrinsic subcellular localization of these enzymes is the ER with some evidence for nuclear envelope localization [50,51]. The current theory has UGTs spanning the ER lumen, with an intra-luminal a-helix anchor and this integral membrane association has prevented the development of a complete UGT crystal structure to date. Several years ago, an elegant partial crystal structure for the UDPGA, C-terminal binding domain was produced [52,53]. As a result, detailed knowledge of the cytosolic tail has been achieved/gained, but there remains much to be elucidated regarding UGT active site(s) structure and mechanisms of glucuronidation, including addressing the problem of latency. A hypothetical model of the enzyme structure with transporters is shown in Figure 3. The UGTs are classified as type I ER proteins, meaning that they are anchored into the ER membrane through a hydrophobic transmembrane domain and the N-terminus is directed toward the lumen of the ER. It has been shown that the N-terminal signal peptide is essential for the translocation of UGTs into the ER membrane [54]. Without this signal peptide, the affinity of the 17 amino acid transmembrane domain and ER membrane would not occur. Another C-terminal stop transfer sequence is also responsible for translocation of the UGT in the ER membrane [50]. Both sequences are vital for UGT function and proper orientation [50,54]. 6.

Latency

Latency in UGT research is an important concept that primarily relates to the accessibility of the co-substrate (and substrates) to the enzyme. Because UGTs are situated on the luminal side of the ER membrane, substrates and co-substrates are not immediately available to the enzyme [50] and must transit the lumen of the ER either by passive


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X-OGA

UDPGA

Nucleoside diphosphatase

NST UDPGA

ATER X-OGA

UDP

UMP


β-Glucuronidase

ER

X-OH UGT dimer

UGT tetramer

Egasyn

Figure 3. Schematic model of ER-localized UGT oligomers, b-glucuronidase and functionally associated transporters. UGTs consist of the N-terminal aglycone (X--OH)-binding half of the monomer and the UDPGA-binding C-terminus with transmembrane segment and cytoplasmic tail (dark). Accumulating evidence suggests that UGTs are functional as dimers in monoglucuronide formation. Two dimers may interact to form a tetramer in diglucuronide formation. The luminal orientation of UGTs and b-glucuronidase requires the action of additional proteins such as NSTs, transporting the cofactor UDPGA to the lumen of the ER, and multiple organic anion transporters in ER membranes (ATER) transporting glucuronides to the cytosol or back into the ER lumen. Figure reproduced with permission from [51]. AT: Anion transporter; ER: Endoplasmic reticulum; NST: Nucleotide sugar transporter; UDP: Uridine diphosphate; UDPGA: Uridine diphosphate glucuronic acid; UGT: Uridine diphosphate glucuronosyltransferase; UMP: Uridine monophosphate; X-OGA: Aglycone conjugated to glucuronic acid.

diffusion or active transport (Figure 3). The active transporter for the co-substrate was identified in 2001 [55]. As yet active transporters for substrates into the luminal ER or of the glucuronides produced out of the ER has been demonstrated biochemically, but specific transporters have not been identified [56,57]. Latency of activity in UGT enzymes has been observed during the course of in vitro studies of metabolism using microsomes. Intact microsomes from mammalian liver exhibit latency and activation of the enzyme in the microsomal system requires chemical or physical disruption of the microsomal membrane [58]. Currently, latency is primarily overcome by using the pore-forming agent alamethicin [59], which allows maximal exposure of co-substrate and substrates to the enzyme. However, detergents or other surfactants that can disrupt the microsomal membrane can also be employed in enzymatic assays. In some cases, these detergents (particularly the rare compound lubrol) offer tight control for revealing latency in vitro. When investigating latency, if fresh intact microsomes are used with a detergent such as Brij 58 or lubrol titrated in, the transport properties across the microsomal membrane can be studied. However, if alamethicin is used the microsome is completely punctured, which allows study of the complete access of substrates and co-substrate to the enzyme protein itself. Carefully designed studies that incorporate both aspects can determine the relative contributions of indirect inhibition or activation of glucuronidation based on substrate and co-substrate availability due to passive diffusion as well as active transport (detergent approach, more physiological) as opposed to direct effects on enzyme catalysis at 954

the level of the protein such as product inhibition or allosterism (alamethicin approach). There are three theories that have been proposed to explain latency. The first is based on the concept that UGTs exist in different conformational states and suggests variable functional parameters depending on the lipid environment of the enzymes [60]. Support for this was advanced when the first experiments demonstrated that purified and recombinant forms of UGT exhibit different substrate profiles, which may (or may not) be attributed to the presence of detergent in the reaction mix altering enzyme conformation [61]. The second hypothesis originates from the analysis of the position (topology) of the active site of UGTs within the lumenal domain of the ER [60]. Called the Compartmentation Hypothesis, this implies that the transport mechanism for the cofactor UDPGA (a hydrophilic molecule) across the ER membrane in vivo, is not active in vitro and thus perturbation of the lipid membrane enables the UDPGA to access the active site of the enzyme. Although all theories are unproven, this second model is generally held to be the most physiologically plausible especially in light of research confirming the presence and activity of a transluminal UDPGA transporter (also called the UST74C transporter) [55,62]. Subsequently, it was demonstrated that multiple nucleotide sugar transporters could transport UDPGA into microsomes prepared from rat liver and a rat hepatoma cell line [63]. Most recently, the assay conditions that contribute to rates and amounts of transport of UDPGA in human liver microsomes have been resolved [56]. The authors demonstrated biphasic transport of UDPGA across the microsomal membrane that contained




both high- and low-affinity components. Coupled with studies where the nucleotide sugar, drugs and glucuronide metabolites in the incubations were varied; the authors report that multiple proteins are likely involved in UDPGA transport, or at least a protein with multiple binding sites [56]. Moreover, based on the inhibition noted, they speculated that some drug and chemical-mediated inhibition of glucuronidation may be through interfering with UDPGA transport [56]. A third potential explanation for latency has recently been advanced [64,65]. Adenine-containing nucleotides can inhibit UGT activity (discussed below), and the concept has been advanced that when the microsomal membrane is perturbed, ATP, NAD+ and NADP+ are released, thereby decreasing natural UGT endogenous inhibition [65]. Indeed recent biochemical evidence seems to point to a role for ATP in the latency of UGTs [64], and this is an interesting idea that needs further confirmation regarding the magnitude and criticality of this putative mechanism’s contribution to latency.

Posttranslational modifications affecting UGT activity

7.

Glycosylation of UGT proteins Glycosylation is likely to be an important mechanism for UGT catalytic activity because it is critical for correct transport to/from the ER [66], in protein folding for UGT1A9 [67] and for catalytic activity of UGT1A and 2B isoforms [68,69]. Asparagine-linked (N-linked) glycosylation is a highly conserved mechanism for the posttranslation transfer of oligosaccharides onto proteins within the ER lumen. Mutations resulting in complete deficiencies of N-glycosylation are lethal in both mammals and yeast. However, minor disruption to the N-glycosylation pathway in humans (such as low-activity genetic variants) can lead to intestinal disorders, liver dysfunction and mental retardation, without overt death [70]. Glycosylation is achieved by transferring a relatively large, lipid-linked oligosaccharide from the ER membrane onto the asparagine residue of the protein consensus sequence, Asn-X-Thr/Ser [71]. This reaction is catalyzed by the membrane bound oligosaccharyl transferase complex. Covalently linked glycans are then available for modification by glycosyl hydrolase and glycosyl transferase accounting for the large variety of glycan structures [71]. The major outcomes of N-glycosylation are effects on protein structure and function [70,71]. For some proteins, glycosylation has a key role in folding and protein stability, but in others glycosylation does not contribute to protein folding [66]. Since UGTs are enzymes, correct folding is vital to produce active and co-factor sites catalysis. There are also indications that glycosylation may be important in associating the protein with the right chaperone proteins or protein oligomers [66]. In proteins where glycosylation is important for folding, loss of a single glycosylation site does not necessarily result in a misfolded protein. Instead, it is the culmination of glycosylation 7.1

sites and interactions that regulate protein folding. Furthermore, in many proteins, once folded, glycosylation is not necessary for maintaining protein conformation [66]. Correct trimming of glycoproteins by glucosidases has key implications for protein transport out of the ER through binding with lectin-like [66,70]. Glycosylation also has extensive effects on protein folding, polymerization and transport, although these are less likely to be important for UGTs since, once made, the vast majority of these enzymes are anchored in the ER (the exception being the UGTs apparently located in the nuclear envelope, which is controversial) [72]. In 2000, Barbier et al. demonstrated the presence of N-glycosylation for human UGT2B7 and UGT2B15 was critical but not for UGT2B4. Moreover, the specific glycosylation site N-96 was the influential residue for UGT2B15 but not UGT2B7 [68]. The UGT2B15 activity decreases were substrate-dependent. Glucuronidation decreased up to 10fold for 3-a-diol, 15-fold for dihydrotestosterone and 4-fold for testosterone. These changes were velocity changes rather than changes to the enzyme’s Km. These authors further demonstrated that enzymatic cleavage of N-linked glycans by endoglycosidase H (Endo H) resulted in reduced human and rat UGT activities. These data show that posttranslational N-linked glycosylation is a regulator of UGT activity [68]. Using similar methods, a study by Nakajima et al. demonstrated that UGTs expressed in HEK293 cells contained N-linked glycans in the variable first exon as well as the conserved two to five exons for the UGT1A1, 1A4 and 1A9 enzymes. Furthermore, this study demonstrated that deglycosylation of UGT1A9 did not yield any functional defects [67]. However, when UGTs are translated in the presence of Tunicamycin, a potent inhibitor of N-glycosylation, UGT1A9 activity for 4-methylumbelliferone was abolished. These results seem to imply that N-glycosylation of UGT1A9 is required for correct folding and activity. Yet, once folded, removal of N-glycans does not result in protein unfolding or loss of function [67]. Most recently, using a HEK293 cell line transformed to express UGT2B7, it was shown that enzymatic deglycosylation of UGT2B7 with EndoH removed one of the three known N-glycosylation sites (N-67), yet N-glycans still remained at two other sites -- N68 and N315 [69]. Functionally, UGT2B7 had a decreased affinity for AZT, but an increased affinity for morphine. This results in up to a fourfold decrease in AZT intrinsic clearance but no changes in morphine-3-O-glucuronidation or morphine-6-O-glucuronidation. Furthermore, deglycosylation resulted in a decreased affinity for UDPGA regardless of the substrate and a decreased consumption of the co-substrate [69]. These authors also generated constructs with mutations at all three known N-glycosylation sites, and the mutants lacked any glucuronidation activity. These results elegantly demonstrated that while the potential glycosylation sites for UGTs are known, which sites are glycosylated and their individual and combined effects on UGT functions are unclear [69].


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Collectively, the published literature implies a significant role for N-linked glycosylation in UGT structure and function. Data from our own laboratory (unpublished, not shown) seem to indicate that N-linked glycosylation occurs in a developmentally regulated manner, and may be involved with ontogenetic switching on UGTs from nonfunctional to active proteins (discussed below). It is clear that the many structural aspects of UGTs have not been elucidated. Therefore, to better understand UGTs and their function in the human for clearance and detoxification, further study of UGT glycosylation -- both N- and O-linked (see below) could be of great benefit in determining environmental and ontogenetic regulation of UGTs. Phosphorylation of UGT proteins Most proteins requiring phosphorylation are multiply phosphorylated. This can occur through the action of a number of different kinases or by one kinase, acting at multiple sites. In the case of enzymes, the protein may require many phosphorylation events to become active [73]. Compared to glycosylation, phosphorylation adds less molecular mass to the protein; however, phosphorylation is a critical regulatory step for many enzymes and enzyme families [73]. The first evidence that phosphorylation is critical for UGT activity came from studies in UGT1A1 transformed COS-1 cells, where it was demonstrated that triple mutants of T75A/T112A/S435G in UGT1A1 (at predicted PKC sites) possessed only 10 -- 15% activity toward bilirubin, while single and double mutants at these sites had varying effects on metabolizing activities [74]. Subsequently, phosphorylation was demonstrated to be critical for the GI-specific isoform UGT1A7 activity [75] as well as UGT1A1, 1A3, 1A4, 1A6, 1A9, 2B7 and 2B15 activities, which were inhibited by dephosphorylation [76]. Using selective inhibitors of various protein kinases, these authors have demonstrated that the major protein kinase of interest for UGT activity is PKCepsilon [75-77]. More recently, it has also been confirmed that a complex pattern of regulated phosphorylation regulates UGT2B15 in the prostate, particularly with relation to maintaining homeostasis with respect to dihydrotestosterone [78]. These authors clearly demonstrated that regulation of UGT2B15 by phosphorylation is determined by both PKCa and Src kinase phosphorylation [78]. Interestingly, while O-linked glycosylation has not yet been confirmed as a posttranscriptional regulatory element for UGT activity, this mechanism competes with serine/ threonine phosphorylation [79]. Because both serine/threonine phosphorylation and N-linked glycosylation are important posttranscriptional regulatory element for UGTs, the role of O-linked glycosylation alone, and with PKC regulation should be pursued. Future studies can begin to address the signaling pathways behind these posttranslational modifications, including the importance of other pathways such as degradation. The turnover rates of UGTs are unknown and mechanisms such as 7.2

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ubiquitination or sumoylation could be important. These involve the binding of small molecules to the proteins, signaling them for proteasomal degradation. Some drug transporters are known to undergo this process, but to date UGTs have not been studied [80]. 8.

Allosteric interactions

The UGT enzymes exhibit allosterism, although the presence and characteristics of precise binding clefts have yet to be definitively identified, primarily because the field lacks a crystal structure and relies on homology models. Thus, allosteric effects tend to be reported in the context of carefully constructed biochemical experiments, which have shown that not all UGT enzymes exhibit Michaelis--Menten kinetics and sigmoidism as well as biphasic kinetics can occur as a consequence of allosteric interactions that are substratedependent [81]. Endogenous compounds The most widely known endogenous interaction that has quasi-regulatory functions on UGT activity at the protein level is the so-called ‘albumin effect’ [82]. Studies performed in vitro with expressed human UGTs have indicated that when added to enzyme incubations, bovine serum albumin (BSA) [83] and fatty acid-free human serum albumin enhance the glucuronidation of UGT1A9 but not UGT1A1 substrates (Table 1) [81]. All forms of albumin alter the kinetic model for 4-methylumbelliferone glucuronidation by UGT1A6 (from Michaelis--Menten to two-site) and BSA has a similar effect on glucuronidation by UGT1A1, but not other forms of albumin [82]. Subsequently, it was demonstrated that BSA decreases the Km for UGT1A9 and 2B7 and increases the Vmax for UGT1A9 toward 4-methylumbelliferone and other substrates [84]. These are important regulatory considerations in vitro and should be used to guide laboratory determinations of UGT disposition, but their relevance to in vivo regulation of glucuronidation is not certain. The generally accepted hypothesis here is that BSA affects UGT catalysis by removing competitive inhibitors, possibly long-chain fatty acids, from the substrate binding site of the enzyme [82,85]. However, recent data demonstrate that instead, the inhibitor (s) that BSA removes binds probably to either enzymeUDPGA or enzyme-UDPGA-substrate complexes rather than to the free enzyme [84]. In addition to this albumin effect, several endogenous substances can regulate UGT activities at the protein level. High levels of ATP, NAD+ and NADP+ can inhibit UGT1A1-mediated formation of estradiol-3-glucuronide in humans although the concentrations tested spanned from physiological to supra-physiological levels [86] and several other species [65]. Speculation regarding how and why adenine nucleotides could inhibit glucuronidation in the ER has subsequently focused on the role(s) of UGTs in steroid hormone metabolism [65]. 8.1



Table 1. Allosteric activators and inhibitors of UGTs. Isoforms


Endogenous Albumin Bovine serum albumin

UGT2B7 UGT1A9 UGT2B7

Substrate in vitro 4MU Entacapone Zidovudine 4MU 4-MU Bilirubin

UDP-xylose UDP-Nacetylglucosamine Acyl-CoA

UGT1A UGT2B

Dihydrotesterone

UGT1A4

4-MU aglycone substrates Tamoxifen

Trans-androsterone

UGT1A4

Tamoxifen

Fatty acids

UGT1A9 UGT2B7

4-MU

Xenobiotic Oxymetazoline

Activation/ inhibition

Enzyme source

Ref.

Activation Activation

rProtein human liver microsomes rProtein human liver microsomes

[82,85] [84]


Rat liver microsomes Guinea-pig and rat liver microsomes

[92] [94]

Activation

Rat liver microsomes

[89]

Activation/ inhibition Activation/ inhibition Inhibition

rProtein

[97]

rProtein

[97]

rProtein Human kidney microsomes

[81]

[98]

UGT1A9

Inhibition

UGT2B7 UGT1A9 UGT2B7 UGT1A9 UGT1A6


Human liver microsomes and recombinant proteins Human and Cynomolgus monkey microsomes rProtein and rat liver microsomes

Activation

rProtein and human liver microsomes

[101]

Oxyresveratrol

Inhibition

[102]

cis-Resveratrol

Inhibition

rProtein and human liver and intestinal microsomes rProtein and human liver microsomes

Activation

rProtein

[104]

Activation Activation Activation Activation Inhibition

Human liver microsomes rProtein Human liver microsomes Human liver microsomes rProtein

[105] [106] [107] [106] [97]

Propanolol Methylenedioxy methamphetamine (Z)-combretastatin A-4

UGT1A1 UGT1A9 UGT1A10 Tricyclic antidepressants UGT1A4 UGT2B10 17b-Estradiol UGT1A1 Propofol UGT1A1 Daidzein UGT1A1 17a-Ethynylestradiol UGT1A1 Lamotrigine UGT1A4

[99] [100]

[103]

rProtein: Recombinant protein; UGT: Uridine diphosphate glucuronosyltransferase.

More study has been devoted to the long-chain fatty acids and their effects on UGTs. In rats, fatty acids inhibit activated microsomal UGTs [87-89]. Specifically, palmitic and oleic acids only inhibit microsomes activated by alamethicin, and it has been suggested that this is not dependent on binding to the active site of the enzyme, but altering the functional state of the UGT enzymes themselves [90]. Additionally, arachidonic acid (in the oleoyl-CoA form) can inhibit UGT activity toward testosterone [88]. In one study, acylation of UGT isoforms by acyl-CoA occurred during in vitro incubation when acyltransferase was not present in the reactions; therefore nonenzymatic auto-acylation has been suggested as part of this mechanism [65]. Interestingly, long-chain fatty acids bind in the C-terminal domain of UGT proteins, which is not the UDPGA-binding site, nor the (generally accepted) active site of the enzyme. This suggests that fatty acids cause

some type of protein conformation change that affects catalysis [65] or that they are binding an allosteric site. It should be noted that conformational changes in UGT proteins as the reason for altering catalysis are controversial. Additionally, there are constraints to intracellular residence of fatty acyl-CoAs. Although they are somewhat amphiphilic and can transit membranes slowly, they are found at much higher concentrations in the cytosol. Therefore, since UGTs are resident in the intra-luminal ER this mechanism would only be relevant in vivo to a lesser extent than seen in vitro and/or if the ER was perturbed, and/or if there are as-yet unknown fatty acid transporters that directly shuttle these compounds across the ER. Several endogenous activators of UGT have been described. Chief among these are other UDP sugars, including UDP-xylose and UDP-N-acetylglucosamine, which


957



appear to be active in a variety of rodent species, and in humans (Table 1) [91-94]. Interestingly, for the UGT1A and 2B subfamilies, addition of UDP-N-acetylglucosamine appears to shift UGT kinetic parameters from a standard Michaelis--Menten model (when UDPGA is used) to a twosite kinetic model of bilirubin glucuronidation [93]. This suggests that UDP-N-acetylglucosamine directly alters UDPGA/ UGT binding. However, an alternative theory based on contra-transport of UDPGA breakdown products and UDPN-acetyl glucosamine that would have a net effect of delivering more UDPGA has also been proposed [91]. Definitive elucidation of these mechanisms has not yet been achieved. In contrast, for UGT3A1 and 3A2, Michaelis--Menten kinetics are observed for UDP-N-acetyl glucosamine, which the enzymes are able to use directly for conjugation [18,27,95], they can also use UDPGA, UDP-galactose and UDP-xylose to varying extents. There also exists a report in the literature that UDP-xylose can stimulate glucuronidation; however, the mechanism for this is believed to be direct facilitation of UDPGA transport rather than a physical interaction with the enzyme [92]. There is also some evidence that negative cooperative kinetics can occur when different UDP-sugars are co-incubated with UGTs. For example, morphine metabolism by UGT2B7 in the presence of UDPGA and UDP-glucose resulted in a 70% lower Clint for morphine3-glucoside than without UDPGA [96]. These in vitro experiments produced metabolite concentrations more relevant to in vivo and suggest that co-incubations with UDP-sugars could be useful in examining inhibition and activation of UGTs [96]. UGT activation by endogenous compounds that are not UDP sugars is less studied. One notable exception is the observation that acyl-CoAs can enhance Vmax, although this results in the concurrent decrease of Km toward the aglycone substrate. Moreover, an increase in Km for UDPGA is coupled with an increase in Vmax toward the aglycone. This latter mechanism is related to the acyl-CoA-related activation of UGT [89]. Furthermore, it has been demonstrated that UGT1A4-mediated glucuronidation of dihydrotestosterone and trans-androsterone follow atypical kinetics with both homo- and heterotropic kinetics occurring due to the presence of multiple binding sites. These studies were performed with UGT1A4-transformed HEK293 cells and the validity of this finding in in vivo as well as its biological relevance remains to be seen [97]. Xenobiotics As compared to endogenous compounds, there are fewer studies where allosterism has been definitively shown for xenobiotics. Both homotropy (multiple site kinetics, where the allosteric binder is also a substrate) and heterotropy (where the allosteric modulator is not also a substrate) have been described for UGTs [98-107]. Autoactivation and inhibition have been observed for both mechanisms as detailed specifically in Table 1. 8.2

958

Another consideration for allosterism is whether all sources of UGT enzyme display the same kinetics -- that is, whether the allosterism noted is source-dependent. This has been covered in review [108]. However, briefly, in at least one published study determining kinetics of UGT1A9 (entacapone) and UGT2B7 (AZT) the effects of BSA on allosterism were source-independent [84]. Namely, the effect of BSA addition on AZT glucuronidation by UGT2B7 and entacapone by UGT1A9 were similar between HLM, recombinant enzymes expressed in insect cell membranes and recombinant enzymes in HEK293 cells [84]. However, these findings were at odds with previous studies that demonstrated differences between recombinant and microsomal kinetics [82,85]. Since there are not standardized assay conditions between laboratories world wide for glucuronidation, these issues are difficult to resolve. Taken together, allosterism is an important regulatory mechanism for glucuronidation of endo- and xenobiotics and appears to be mediated in both homotropic and heterotropic manner that is enzyme and substrate-dependent. The relevance and translation of in vitro studies where allosterism is demonstrated, to in vivo glucuronidation and clinical outcomes is an ongoing area of interest. One potential area where allosterism could be of clinical concern is in polypharmacy -- namely when a patient is taking multiple drugs that may interact allosterically at the same UGT isoenzyme. Since both activation and inhibition of UGTs by allosteric modulators have been observed atypical, subtherapeutic or toxic responses to drugs may occur. This is particularly relevant now that the pharmaceutical industry is targeting conjugation as the preferred pathways of metabolism, because other concurrent pathways of metabolism (that confer protective redundancy) are designed out of drug. Where metabolism, is by a single enzyme isoforms and/or families, these considerations will become increasingly relevant.

Intra- and inter-family protein--protein interactions

9.

Protein--protein interactions between UGTs and other metabolizing enzymes (notably CYP450) as well as dimerization and oligomerization within the UGT subfamily have been proposed to alter glucuronidation [109-114]. Specifically, interactions between UGTs and CYP450s and well as homoand heterodimerization have been demonstrated to increase glucuronidation capacity and affect substrate selectivity. The first evidence for protein--protein interactions of UGTs came from Gunn rats where it was demonstrated that different proteins from the UGT1 family could be co-immunoprecipitated with UGT2B1 proteins and that functionally, the uptake of UDPGA could be affected by formation of these complexes [115]. In humans, this was first demonstrated by the coimmunoprecipitation of UGT with CYP450 1A1 that was deemed to be selective since disulfide isomerase and calnexin from the same preparations did not coprecipitate [114].




Subsequently, the potential for UGT oligomerization was deduced by radiation inactivation where it was shown that UGT1A1 probably acts as a homodimer, although trimers or even tetramers are possible conformations [109]. This was followed by specific demonstration that UGT1A6, 1A1 and 2B7 could co-immunoprecipitate with each other and with CYP3A4, leading the authors to speculate that protein--protein interactions of UGTs could be within the superfamily (forming dimers, tetramers, etc.) as well as between drug-metabolizing families and affect total metabolism [72]. Subsequently, Operana and Tukey used both co-immunoprecipitation and fluorescence resonance energy transfer experiments to show specifically that UGT1A1, UGT1A3, UGT1A4, UGT1A6, UGT1A7, UGT1A8, UGT1A9 and UGT1A10 can homodimerize, while UGT1A1 can heterodimerize with UGT1A3, UGT1A4, UGT1A6, UGT1A7, UGT1A8, UGT1A9 and UGT1A10, which the authors suggested could affect enzyme function and substrate specificity [112]. This was quickly confirmed by Fujiwara et al. who used double expression systems of UGT1A1/UGT1A4, UGT1A1/1A6 and UGT1A4/1A6 to demonstrate that these isoforms interact with each other, with complex effects on enzymatic activities that were isoform and substrate-dependent, but could alter both Vmax and Km [116]. These authors followed up by demonstrating that these interactions were not limited within the subfamily and that coexpression of UGT2B7/UGT1A1, UGT2B7/UGT1A4, UGT2B7/UGT1A6 and UGT2B7/UGT1A9 all increased the thermostability of the UGT2B7 isoform and altered metabolism of zidovudine (primarily mediated by UGT2B7) as well as metabolism (Km and Vmax) of specific substrates for each UGT1A isoenzyme [117]. It has been shown that the complex-forming phenomenon that affects enzyme function goes beyond UGT/UGT complexes, since studies in rats have demonstrated that CYP3A2, CYP2B2, CYP2C11/13 and CYP1A2 coprecipitate with UGTs and these complexes are functional toward the pan-specific substrate 4-methylumbelliferone with differing rates of metabolism [110]. UGT1A1 has reported to be closely associated with UGT1A6 as well as UGT2B7 in native ER membranes helping to validate its capacity for heterodimerization [111]. More recently, through co-immunoprecipitaiton, it has been shown that UGT2B7 acts as a homodimer. Functionally, it has been hypothesized that oligomeric UGT-binding sites act as one in substrate glucuronidation [118]. Current approaches to mass spectrometry detection of UGT proteins and multiplexed proteomics have great promise to illuminate these phenomena [119,120]. 10.

Ontogenetic regulation of UGT

Developmental absence of drug-metabolizing enzymes is often quoted as the reason for adverse drug reactions. While this is not always true, the UGT enzymes are an example of a critical drug elimination pathway that does not mature until after birth [36-39,121-126]. Although UGTs can be detected as

early as the blastocyst stage in mammalian development (indicating a possible role in embryology) [127] in fetal red blood cells and in the fetal liver [128-130], glucuronidation activity has not been detected until 30 -- 40 weeks of gestation [122,123]. This is not due to a lack of UDPGA co-substrate, because the human fetal liver and kidney as well as the placenta contain UDPGA at the same concentrations as adults [131]. At birth, glucuronidation is around 1% of adult levels, with continuing post-natal maturation related to birth age not gestational age [122]. Subsequent biochemical studies progressively delineated which UGT isoforms were present and active [128,132] culminating in the first isoform-specific studies from Strassburg et al. who showed that although fetal levels of mRNA and protein for UGT isoforms did not differ from adults, activities were lower for up to 2 years of age [36]. Subsequently, the pediatric ontogeny of several hepatic UGT1A and UGT2B isoforms has been described [36-39,121-126] although the development of UGT enzymes in other neonatal tissues is not well understood. The liver studies have demonstrated that although mRNA and protein are present, glucuronidation does not mature for several months after birth, with maturation of individual UGT enzymes each under separate developmental controls [37-39,121,126]. As noted above (Section 4), in addition to inactivity of the proteins that are present, there are lower levels of UGT proteins in fetuses and neonates (up to 2 years old) as compared to adults [36,40]. Moreover, liver capacity for drug elimination through UGT pathways is a combination of biochemical and physiological development, and takes years to develop [37-39]. Because the proteins are present but inactive, a posttranslational regulation has been speculated as the mechanism for the developmental switch. Whether this occurs through endogenous molecule effects on the protein (such as changes in the levels of inhibitory or activating endogenous molecules) or through direct protein modification (such as through glycosylation or phosphorylation) is not known at this time, although preliminary data from our laboratories indicates that N-linked glycosylation is a potential mechanism (data not shown). This would be empirically sensible because glycosylases are also developmentally regulated, and congenital defects in these enzymes cause and/or contribute to developmental delay, hepatic fibrosis, hepatic steatosis, protein-losing enteropathy, coagulopathy, hypoglycemia and failure to thrive [133]. Moreover, glycosylation is not ‘template driven’ in the way that transcription and translation are, and both genetic and environmental factors may interact to influence glycosylation mechanisms [134]. 11.

Conclusion

There is no doubt as to the plasticity of UGTs, and their responsiveness at the transcriptional level. However, posttranscriptional regulation of glucuronidation is less studied and is not well understood. The current literature demonstrates many effects of endo- and xenobiotics directly on


959



glucuronidation, which includes both homo- and heterotropic activation as well as autoinhibition. Moreover, indirect effects of compounds that affect glucuronidation rates and/or capacity such as inhibition of UDPGA transport may have regulatory functions for the enzymes and these data are emerging, primarily from in vitro studies [56,64]. Classically, some endoand xenobiotics have been shown to alter glucuronidation at levels that could be dietarily and/or environmentally relevant and, while many times these occur through competitive inhibition of UGT activity competitive enzyme kinetics have not been covered comprehensively here. A critical need in the field is identification of isoform-specific inhibitors of UGTs for reaction phenotyping. Evidence to date suggests that UGT enzymes can form homo- and hetero-complexes within the UGT family and consensus appears to be that these are either dimers or tetramers, but full molecular elucidation of this has not yet been achieved. Moreover, there is evidence that UGTs can form complexes and act cooperatively at the protein level with other enzyme families, notably the CYP450s. Again, the molecular and functional mechanisms of these interactions are not yet elucidated and their in vivo relevance has not been clearly established. 12.

Declaration of interest

Expert opinion

Although several posttranscriptional regulatory mechanisms have been described herein, often it is not possible to determine if the endogenous compound effects observed in vitro at the posttranslational level truly occur in vivo, or whether they are artifacts of experimentation. This is particularly relevant in relation to access of cytosolic compounds to UGT Bibliography Papers of special note have been highlighted as either of interest () or of considerable interest () to readers. 1.

Dutton G. Glucuronidation of drugs and other compounds. CRC Press; Boca Raton, FL: 1980

2.

Mackenzie PI, Bock KW, Burchell B, et al. Nomenclature update for the mammalian UDP glycosyltransferase (UGT) gene superfamily. Pharmacogenet Genomics 2005;677-85

3.

Radominska-Pandya A, Czernik PJ, Little JM, et al. Structural and functional studies of UDP-glucuronosyl transferases. Drug Metab Rev 1999;31:817-99

4.

Tukey RH, Strassburg CP. Human UDP-glucuronosyltransferases: metabolism, expression, and disease. Ann Rev Pharmacol Toxicol 2000;40:581-616

960

enzymes on the luminal side of the ER. A return to the use of detergents to reveal latency, in addition to experiments with alamethicin can begin to resolve this biochemically and progress in the field of transport proteins, to understand the balance between diffusion and transport, will also elucidate these issues. Perhaps the most encouraging potential mechanism for posttranscriptional regulation of UGTs is posttranslational modification of UGT protein structure, such as by glycosylation and/or phosphorylation, as a regulatory control on catalysis. This is also a promising mechanism for determining the ‘developmental switch,’ by which dormant UGT proteins become activated in the neonatal liver. These latter mechanisms are difficult to study as they require delicate and selective manipulation of ribosomal and ER functions as well as UGT proteins. These are technically demanding experiments. While classical biochemical approaches continue to bear fruit for determining active-site interactions as well as allosteric modulation, understanding of the mechanisms for posttranscriptional regulation of UGTs will be greatly improved once a defined crystal structure is achieved, and as technology advances in the area of proteomics.

5.

The authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties.

Sallustio BC. Acyl Glucuronides as toxic intermediates. In: Hodgson WC, Lioacono RE, editors. Australasian Society for Clininal and Experimental Pharmacology and Toxicology. 2000 December 2000 ASCEPT society; Newcastle, Australia: 2000. p. 60

6.

Court M. Isoform selective probe substrates for in vitro studies of human UDP-glucuronosyltransferases. Methods Enzymol 2005;400:104-16

7.

Tukey R, Strassburg C, Mackenzie P. Pharmacogenomics of human UDPglucuronosyltransferases and irinotecan toxicity. Mol Pharmacol 2002;62:446-50

8.

Evans WE, Relling M. Pharmacogenomics: translating functional genomics into rational therapeutics. Science 1999;286:487-91

9.

Burchell B, Brierley CH, Rance D. Specificity of human UDPglucuronosyltransferases and xenobiotic


glucuronidation. Life Sci 1995;57:1819-31 10.

Guillemette C, Levesque E, Harvey M, et al. UGT genomic diversity: beyond gene duplication. Drug Metab Rev 2010;42:24-44

11.

Hu D, Meech R, McKinnon R, et al. Transcriptional regulation of human UDP-glucuronosyltransferase genes. Drug Metab Rev 2014;46:421-58

12.

Bock K. Human UDPglucuronosyltransferases: feedback loops between substrates and ligands of their transcription factors. Biochem Pharmacol 2012;84:1000-6

13.

Bock K. Functions and transcriptional regulation of adult human hepatic UDP-glucuronosyl-transferases (UGTs): mechanisms responsible for interindividual variation of UGT levels. Biochem Pharmacol 2010;80:771-7


14.

.


15.

.

16.

17.

Yasar U, Greenblatt DJ, Guillemette C, et al. Evidence for regulation of UDP-glucuronosyltransferase (UGT) 1A1 protein expression and activity via DNA methylation in healthy human livers. J Pharm Pharmacol 2013;65:874-83 First identification of epigenetic modification of UGTs. Oda S, Fukami T, Yokoi T, et al. Epigenetic regulation is a crucial factor in the repression of UGT1A1 expression in the human kidney. Drug Metab Dispos 2013;41:1738-43 First identification of epigenetic modification of UGTs. Oda S, Fukami T, Yokoi T, et al. Epigenetic regulation of the tissuespecific expression of human UDPglucuronosyltransferase (UGT) 1A10. Biochem Pharmacol 2014;87:660-7 Mackenzie PI, Hu DG, Gardner-Stephen DA. The regulation of UDP-glucuronosyltransferase genes by tissue-specific and ligand-activated transcription factors. Drug Metab Rev 2010;42:99-109

18.

Meech R, Mackenzie PI. UGT3A: novel UDP-glycosyltransferases of the UGT superfamily. Drug Metab Rev 2010;42:45-54

19.

Rowland A, Miners JO, Mackenzie PI. The UDP-glucuronosyltransferases: their role in drug metabolism and detoxification. Int J Biochem Cell Biol 2013;45:1121-32

20.

Gong Q, Cho J, Huang T, et al. Thirteen UDPglucuronosyltransferase genes are encoded at the human UGT1 gene complex locus. Pharmacogenetics 2001;11:357-68

21.

Caspersen C, Reznik B, Weldy P, et al. Molecular cloning of the baboon UDPglucuronosyltransferase 1A gene family: evolution of the primate UGT1 locus and relevance for models of human drug metabolism. Pharmacogenet Genomics 2007;17:11-24

24.

25.

26.

27.

28.

Mackenzie PI, Gregory PA, Lewinsky RH, et al. Polymorphic variations in the expression of the chemical detoxifying UDP glucuronosyltransferases. Toxicol Appl Pharmacol 2005;207:S77-83 Sneitz N, Court M, Zhang X, et al. Human UDP-glucuronosyltransferase UGT2A2: cDNA construction, expression, and functional characterization in comparison with UGT2A1 and UGT2A3. Pharmacogenet Genomics 2009;19:923-34 Bushey RT, Dluzen D, Lazarus P. Importance of UDPglucuronosyltransferases 2A2 and 2A3 in tobacco carcinogen metabolism. Drug Metab Dispos 2013;41:170-9 Mackenzie PI, Rogers A, Treloar J, et al. Identification of UDP glycosyltransferase 3A1 as a UDP N-acetylglucosaminyltransferase. J Biol Chem 2008;283:36205-10 MacKenzie P, Rogers A, Elliot D, et al. The novel UDP glycosyltransferase 3A2: cloning, catalytic properties, and tissue distribution. Mol Pharmacol 2011;79:472-8

29.

Meech R, Rogers A, Zhuang L, et al. Identification of residues that confer sugar selectivity to UDPglycosyltransferase 3A (UGT3A) enzymes. J Biol Chem 2012;287:24122-30

30.

Bosio A, Binczek E, Le Beau M, et al. The human gene CGT encoding the UDP-galactose ceramide galactosyl transferase (cerebroside synthase): cloning, characterization, and assignment to human chromosome 4, band q26. Genomics 1996;34:69-75

31.

Meech R, Mubarokah N, Shivasami A, et al. A novel function for UDP glycosyltransferase 8: galactosidation of bile acids. Mol Pharmacol 2015;87:442-50

22.

Owens I, Basu N, Banerjee R. UDPglucuronosyltransferases: gene structures of UGT1 and UGT2 families. Methods Enzymol 2005;400:1-22

32.

Bock K. Vertebrate UDPglucuronosyltransferases: functional and evolutionary aspects. Biochem Pharmacol 2003;66:691-6

23.

Mackenzie PI, Gregory PA, Gardner-Stephen DA, et al. Regulation of UDP glucuronosyltransferase genes. Curr Drug Metab 2003;4:249-57

33.

Shrestha B, Reed J, Starks P, et al. Evolution of a major drug metabolizing enzyme defect in the domestic cat and other felidae: phylogenetic timing and the role of hypercarnivory. PLoS One 2011;6:e18046


34.

McCarroll S, Hadnott T, Perry G, et al. Common deletion polymorphisms in the human genome. Nat Genet 2006;38:86-92

35.

Izukawa T, Nakajima M, Fujiwara R, et al. Quantitative analysis of UDPglucuronosyltransferase (UGT) 1A and UGT2B expression levels in human livers. Drug Metab Dispos 2009;37:1759-68

36.

Strassburg C, Strassburg A, Kneip S, et al. Developmental aspects of human hepatic drug glucuronidation in young children and adults. Gut 2002;50:259-65

37.

Miyagi SJ, Collier AC. Pediatric development of glucuronidation: the ontogeny of hepatic UGT1A4. Drug Metab Dispos 2007;35:1587

38.

Miyagi SJ, Collier AC. The development of UDP-glucuronosyltransferases 1A1 and 1A6 in the pediatric liver. Drug Metab Dispos 2011;39:912-19

39.

Miyagi SJ, Milne AM, Coughtrie M, et al. The neonatal development of hepatic UGT1A9: implications of pediatric pharmacokinetics. Drug Metab Dispos 2012;40:1321-7

40.

Sato Y, Nagata M, Tetsuka K, et al. Optimized methods for targeted peptidebased quantification of human uridine 5’-diphosphate-glucuronosyltransferases in biological specimens using liquid chromatography-tandem mass spectrometry. Drug Metab Dispos 2014;42:885-9

41.

Hardwick RN, Ferreira DW, More VR, et al. Altered UDPglucuronosyltransferase and sulfotransferase expression and function during progressive stages of human nonalcoholic fatty liver disease. Drug Metab Dispos 2013;41(3):554-61

42.

Strassburg CP, Kneip S, Topp J, et al. Polymorphic gene regulation and interindividual variation of UDPglucuronosyltransferase activity in human small intestine. J Biol Chem 2000;275(46):36164-71

43.

Gregory PA, Lewinsky RH, Gardner-Stephen DA, PI M. Regulation of UDP glucuronosyltransferases in the gastrointestinal tract. Toxicol Appl Pharmacol 2004;199(3):354-63

44.

Fisher MB, Paine MF, Strelevitz TJ, SA W. The role of hepatic and extrahepatic UDP-

961


glucuronosyltransferases in human drug metabolism. Drug Metab Rev 2001;33(3-4):273-97 45.


46.

47.

48.

49.

50.

51.

52.

53.

962

Ekstr€ om L, Johansson M, Rane A. Tissue distribution and relative gene expression of UDP-glucuronosyltransferases (2B7, 2B15, 2B17) in the human fetus. Drug Metab Dispos 2013;41(2):291-5 Grosse L, Paˆquet S, Caron P, et al. Androgen glucuronidation: an unexpected target for androgen deprivation therapy, with prognosis and diagnostic implications. Cancer Res 2013;73(23):6963-71 Gauthier-Landry L, Belanger A, Barbier B. Multiple roles for UDP-glucuronosyltransferase (UGT) 2B15 and UGT2B17 enzymes in androgen metabolism and prostate cancer evolution. J Steroid Biochem Mol Biol 2015;145:187-92 Hum DW, Belanger A, Levesque E, et al. Characterization of UDPglucuronosyltransferases active on steroid hormones. J Steroid Biochem Mol Biol 1999;69(1-6):413-23 Turgeon D, Carrier JS, Levesque E, et al. Relative enzymatic activity, protein stability, and tissue distribution of human steroid-metabolizing UGT2B subfamily members. Endocrinology 2001;142(2):778-87 Ouzzine M, Magdalou J, Burchell B, Fournel-Gigleux S. An internal signal sequence mediates the targeting and retention of the human UDPglucuronosyltransferase 1A6 to the endoplasmic reticulum. J Biol Chem 1999;274(44):31401-9 Bock K, Kohle C. Topological aspects of oligomeric UDP-glucuronosyltransferases in endoplasmic reticulum membranes: advances and open questions. Biochem Pharmacol 2009;77(9):1458-65 Radominska-Pandya A, Bratton SM, Redinbo MR, Miley MJ. The crystal structure of human UDPglucuronosyltransferase 2B7 C-terminal end is the first mammalian UGT target to be revealed: the significance for human UGTs from both the 1A and 2B families. Drug Metab Rev 2010;42(1):133-44 Miley M, Zielinska A, Keenan J, et al. Crystal structure of the cofactor-binding domain of the human phase II drugmetabolism enzyme UDP-

glucuronosyltransferase 2B7. J Mol Biol 2007;369(2):498-511 First parietal crystal structure.

.

54.

.

55.

.

56.

57.

.

63.

Kobayashi T, Sleeman JE, Coughtrie MW, et al. Molecular and functional characterization of microsomal UDP-glucuronic acid uptake by members of the nucleotide sugar transporter (NST) family. Biochem J 2006;400:281-9

64.

Ishii Y, An K, Nishimura Y, et al. ATP serves as an endogenous inhibitor of UDP-glucuronosyltransferase (UGT): a new insight into the latency of UGT. Drug Metab Dispos 2012;40:2081-9

Goto S, Taniguchi M, Muraoka M, et al. UDP-sugar transporter implicated in glycosylation and processing of Notch. Nat Cell Biol 2001;3(9):816-22 Discovery and characterization of UDPGA transporter.

65.

Ishii Y, Nurrochmad A, Yamada H. Modulation of UDPglucuronosyltransferase activity by endogenous compounds. Drug Metab Pharmacokinet 2010;25:134-48

Rowland A, Mackenzie P, Miners J. Transporter-mediated uptake of UDPglucuronic acid by human liver microsomes: assay conditions, kinetics, and inhibition. Drug Metab Dispos 2015;43:147-53

66.

Imperiali B, O’Connor S. Effect of N-linked glycosylation on glycopeptide and glycoprotein structure. Curr Opin Chem Biol 1999;3:643-9

67.

Nakajima M, Koga T, Sakai H, et al. N-Glycosylation plays a role in protein folding of human UGT1A9. Biochem Pharmacol 2010;79:1165-72

68.

Barbier O, Girard C, Breton R, et al. N-glycosylation and residue 96 are involved in the functional properties of UDP-glucuronosyltransferase enzymes. Biochemistry 2000;39:11540-52

69.

Nagaoka K, Hanioka N, Ikushiro S, et al. The effects of N-glycosylation on the glucuronidation of zidovudine and morphine by UGT2B7 expressed in HEK293 cells. Drug Metab Pharmacokinet 2012;27:388-97

70.

Freeze H, Westphal V. Balancing N-linked glycosylation to avoid disease. Biochimie 2001;83:791-9

71.

Freeze H. Understanding human glycosylation disorders: biochemistry leads the charge. J Biol Chem 2013;288:6936-45

72.

Fremont J, Wang R, King CD. Coimmunoprecipitation of UDPglucuronosyltransferase isoforms and cytochrome P450 3A4. Mol Pharmacol 2005;67:260-2

73.

Ferrell J, Ha S. Ultrasensitivity part II: multisite phosphorylation, stoichiometric inhibitors, and positive feedback. Trends Biochem Sci 2014;39:556-69

74.

Basu N, Kole L, Owens I. Evidence for phosphorylation requirement for human bilirubin UDP-glucuronosyltransferase

Ouzzine M, Magdalou J, Burchell B, Fournel-Gigleux S. Expression of a functionally active human hepatic UDPglucuronosyltransferase (UGT1A6) lacking the N-terminal signal sequence in the endoplasmic reticulum. FEBS Lett 1999;454(3):187-91 ER trafficking of UGTs.

Revesz K, To´th B, Staines AG, et al. Luminal accumulation of newly synthesized morphine-3-glucuronide in rat liver microsomal vesicles. Biofactors 2013;39:271-8 Discovery and characterization of UDPGA transporter.

58.

Gueraud F, Paris A. Glucuronidation: a dual control. Gen Pharmacol 1998;31:683-8

59.

Fisher MB, Campanale K, Ackermann BL, et al. In vitro glucuronidation using human liver microsomes and the pore-forming peptide alamethicin. Drug Metab Dispos 2000;28:560-6 Established alamethicin as standard for activation since 2000.

.

60.

Zakim D, Dannenberg A. How does the microsomal membrane regulate UDPglucuronosyltransferases? Biochem Pharmacol 1992;43:1385-93

61.

Meech R, Mackenzie P. Structure and function of uridine diphosphate glucuronosyltransferases. Clin Exp Pharmacol Physiol 1997;24:907-15

62.

Selva E, Hong K, Baeg G, et al. Dual role of the fringe connection gene in both heparan sulphate and fringedependent signalling events. Nat Cell Biol 2001;3:809-15 Discovery and characterization of UDPGA transporter.

.



transferred to newly released extracellular vesicles. Transfusion 2013;53:612-26


(UGT1A1) activity. Biochem Biophys Res Commun 2003;303:98-104 75.

Basu N, Kovarova M, Garza A, et al. Phosphorylation of a UDPglucuronosyltransferase regulates substrate specificity. Proc Natl Acad Sci USA 2005;102:6285-90

76.

Basu N, Kole L, Basu M, et al. The major chemical-detoxifying system of UDP-glucuronosyltransferases requires regulated phosphorylation supported by protein kinase C. J Biol Chem 2008;283:23048-61

77.

.

78.

79.

80.

81.

82.

.

83.

Basu NK, Kole L, Owens IS. Evidence for phosphorylation requirement for human bilirubin UDPglucuronosyltransferase (UGT1A1) activity. Biochem Biophys Res Commun 2003;303(1):98-104 First evidence for phosphorylation as a regulatory mechanism. Chakraborty S, Basu N, Jana S, et al. Protein kinase Calpha and Src kinase support human prostate-distributed dihydrotestosterone-metabolizing UDPglucuronosyltransferase 2B15 activity. J Biol Chem 2012;287:24387-96 Vaidyanathan K, Durning S, Wells L. Functional O-GlcNAc modifications: implications in molecular regulation and pathophysiology. Crit Rev Biochem Mol Biol 2014;49:140-63 Zhang Z, Wu J, Hait W, et al. Regulation of the stability of P-glycoprotein by ubiquitination. Mol Pharmacol 2004;66:395-403 Tsoutsikos P, Miners J, Stapleton A, et al. Evidence that unsaturated fatty acids are potent inhibitors of renal UDPglucuronosyltransferases (UGT): kinetic studies using human kidney cortical microsomes and recombinant UGT1A9 and UGT2B7. Biochem Pharmacol 2004;67:191-9 Rowland A, Elliot DJ, Knights KM, et al. The “albumin effect” and in vitro-in vivo extrapolation: sequestration of long-chain unsaturated fatty acids enhances phenytoin hydroxylation by human liver microsomal and recombinant cytochrome P450 2C9. Drug Metab Dispos 2008;36:870-7 Critical role of albumin on catalysis. Pienimaeki-Roemer A, Ruebsaamen K, Boettcher A, et al. Stored platelets alter glycerophospholipid and sphingolipid species, which are differentially

84.

Manevski N, Moreolo P, Yli-Kauhaluoma J, et al. Bovine serum albumin decreases Km values of human UDP-glucuronosyltransferases 1A9 and 2B7 and increases Vmax values of UGT1A9. Drug metab Dispos 2011;39:2117-29

85.

Rowland A, Gaganis P, Elliot DJ, et al. Binding of inhibitory fatty acids is responsible for the enhancement of UDP-glucuronosyltransferase 2B7 activity by albumin: implications for in vitro-in vivo extrapolation. J Pharmacol Exp Ther 2007;321:137-47

93.

Hauser S, Ransil B, Ziurys J, et al. Interaction of uridine 5’diphosphoglucuronic acid with microsomal UDP-glucuronosyltransferase in primate liver: the facilitating role of uridine 5’-diphospho-Nacetylglucosamine. Biochim Biophys Acta 1988;967:141-8

94.

Pogell B, Leloir L. Nucleotide activation of liver microsomal glucuronidation. J Biol Chem 1961;236:293-8

95.

MacKenzie PI, Rogers A, Elliot DJ, et al. The novel UDP glycosyltransferase 3A2: cloning, catalytic properties, and tissue distribution. Mol Pharmacol 2011;79:472-8

86.

Nishimura Y, Maeda S, Ikushiro S, et al. Inhibitory effects of adenine nucleotides and related substances on UDPglucuronosyltransferase: structure- effect relationships and evidence for an allosteric mechanism. Biochim Biophys Acta 2007;1770:1557-66

96.

Chau N, Elliot D, Lewis B, et al. Morphine glucuronidation and glucosidation represent complementary metabolic pathways that are both catalyzed by UDP-glucuronosyltransferase 2B7: kinetic, inhibition, and molecular modeling studies. J Pharmacol Exp Ther 2014;349(1):126-37

87.

Csala M, Banhegyi G, Kardon T, et al. Inhibition of glucuronidation by an acyl-CoA-mediated indirect mechanism. Biochem Pharmacol 1996;52:1127-31

97.

88.

Yamashita A, Nagatsuka T, Watanabe M, et al. Inhibition of UDPglucuronosyltransferase activity by fatty acyl-CoA. Kinetic studies and structureactivity relationship. Biochem Pharmacol 1997;53:561-70

Zhou J, Tracy T, Remmel RP. Glucuronidation of dihydrotestosterone and trans-androsterone by recombinant UDP-glucuronosyltransferase (UGT) 1A4: evidence for multiple UGT1A4 aglycone binding sites. Drug Metab Dispos 2010;38:431-40

98.

Mahajan M, Uttamsingh V, Gan L, et al. Identification and characterization of oxymetazoline glucuronidation in human liver microsomes: evidence for the involvement of UGT1A9. J Pharm Sci 2011;100:784-93

99.

Hanioka N, Hayashi K, Shimizudani T, et al. Stereoselective glucuronidation of propranolol in human and cynomolgus monkey liver microsomes: role of human hepatic UDP-glucuronosyltransferase isoforms, UGT1A9, UGT2B4 and UGT2B7. Pharmacology 2008;82:293-303

89.

Okamura K, Ishii Y, Ikushiro S, et al. Fatty acyl-CoA as an endogenous activator of UDP-glucuronosyltransferases. Biochem Biophys Res Commun 2006;345:1649-56

90.

Krcmery M, Zakim D. Effects of oleoylCoA on the activity and functional state of UDP-glucuronosyltransferase. Biochem Pharmacol 1993;46:897-904

91.

Bossuyt X, Blanckaert N. Mechanism of stimulation of microsomal UDPglucuronosyltransferase by UDP-N-acetylglucosamine. Biochem J 1995;305:321-8 Critical role of co-substrates.

.

92.

Bossuyt X, Blanckaert N. Uridine diphosphoxylose enhances hepatic microsomal UDP-glucuronosyltransferase activity by stimulating transport of UDPglucuronic acid across the endoplasmic reticulum membrane. Biochem J 1996;315:189-93


100. Schwaninger A, Meyer M, Zapp J, et al. The role of human UDPglucuronyltransferases on the formation of the ethylenedioxymethamphetamine (ecstasy) phase II metabolites R- and S-3-ethoxymethamphetamine 4-O-glucuronides. Drug Metab Dispos 2009;37:2212-20 101. Aprile S, Del Grosso E, Grosa G. Identification of the human UDPglucuronosyltransferases involved in the glucuronidation of combretastatin A-4. Drug Metab Dispos 2010;38:1141-6

963



102. Hu N, Mei M, Ruan J, et al. Regioselective glucuronidation of oxyresveratrol, a natural hydroxystilbene, by human liver and intestinal microsomes and recombinant UGTs. Drug Metab Pharmacokinet 2014;29:229-36

111. Lewis BC, Mackenzie PI, Miners JO. Homodimerization of UDPglucuronosyltransferase 2B7 (UGT2B7) and identification of a putative dimerization domain by protein homology modeling. Biochem Pharmacol 2011;82:2016-23

103. Iwuchukwu O, Nagar S. Cis-resveratrol glucuronidation kinetics in human and recombinant UGT1A sources. Xenobiotica 2010;40:102-8

112. Operan˜a T, Tukey R. Oligomerization of the UDP-glucuronosyltransferase 1A proteins: homo- and heterodimerization analysis by fluorescence resonance energy transfer and co-immunoprecipitation. J Biol Chem 2007;282:4821-9 . Critical study on oligomerization.

104. Zhou D, Guo J, Linnenbach A, et al. Role of human UGT2B10 in N-glucuronidation of tricyclic antidepressants, amitriptyline, imipramine, clomipramine, and trimipramine. Drug Metab Dispos 2010;38:863-70 105. Williams J, Ring B, Cantrell V, et al. Differential modulation of UDPglucuronosyltransferase 1A1 (UGT1A1)catalyzed estradiol-3-glucuronidation by the addition of UGT1A1 substrates and other compounds to human liver microsomes. Drug Metab Dispos 2002;30:1266-73 106. Mano Y, Usui T, Kamimura H. Effects of beta-estradiol and propofol on the 4-methylumbelliferone glucuronidation in recombinant human UGT isozymes 1A1, 1A8 and 1A9. Biopharm Drug Dispos 2004;25:339-44 107. Pfeiffer E, Treiling C, Hoehle S, et al. Isoflavones modulate the glucuronidation of estradiol in human liver microsomes. Carcinogenesis 2005;26:2172-8 108. Radominska-Pandya A, Bratton S, Little JM. A historical overview of the heterologous expression of mammalian UDP-glucuronosyltransferase isoforms over the past twenty years. Curr Drug Metab 2005;6:141-60 109. Ghosh S, Sappa LB, Kalpana G, et al. Homodimerization of human bilirubin-uridine-diphosphoglucuronate glucuronosyltransferase-1 (UGT1A1) and its functional implications. J Biol Chem 2001;276:42108-15 110. Ishii Y, Iwanaga M, Nishimura Y, et al. Protein-protein interactions between rat hepatic cytochromes P450 (P450s) and UDP-glucuronosyltransferases (UGTs): evidence for the functionally active UGT in P450-UGT complex. Drug Metab Pharmacokinet 2007;22:367-76 . Identifies functional protein-protein complexes.

964

113. Takeda S, Ishii Y, Iwanaga M, et al. Modulation of UDPglucuronosyltransferase function by cytochrome P450: evidence for the alteration of UGT2B7-catalyzed glucuronidation of morphine by CYP3A4. Mol Pharmacol 2005;67:665-72 114. Taura KI, Yamada H, Hagino Y, et al. Interaction between cytochrome P450 and other drug-metabolizing enzymes: evidence for an association of CYP1A1 with microsomal epoxide hydrolase and UDP-glucuronosyltransferase. Biochem Biophys Res Commun 2000;273:1048-52 115. Ikushiro S, Emi Y, Iyanagi T. Protein-protein interactions between UDP-glucuronosyltransferase isozymes in rat hepatic microsomes. Biochemistry 1997;36:7154-61 116. Fujiwara R, Nakajima M, Yamanaka H, et al. Interactions between human UGT1A1, UGT1A4, and UGT1A6 affect their enzymatic activities. Drug Metab Dispos 2007;35:1781-7 117. Fujiwara R, Nakajima M, Oda S, et al. Interactions between human UDPglucuronosyltransferase (UGT) 2B7 and UGT1A enzymes. J Pharm Sci 2010;99:442-54 118. Finel M, Kurkela M. The UDPglucuronosyltransferases as oligomeric enzymes. Curr Drug Metab 2008;9:70-6 119. Achour B, Russell M, Barber J, et al. Simultaneous quantification of the abundance of several cytochrome P450 and uridine 5’-diphospho-glucuronosyltransferase enzymes in human liver microsomes using multiplexed targeted proteomics. Drug Metab Dispos 2014;42:500-10


120. Fallon J, Neubert H, Hyland R, et al. Targeted quantitative proteomics for the analysis of 14 UGT1As and -2Bs in human liver using Nano UPLC-MS/MS with selected reaction monitoring. J Proteome Res 2013;12:4402-13 121. Divakaran K, Hines R, McCarver D. Human hepatic UGT2B15 developmental expression. Toxicol Sci 2014;14:292-9 122. Kawade N, Onishi S. The prenatal and postnatal development of UDPglucuronyltransferase activity towards bilirubin and the effect of premature birth on this activity in human liver. Biochem J 1981;196:257-60 . Seminal in development. 123. Onishi S, Kawade N, Itoh S, et al. Postnatal development of uridine diphosphate glucuronyltransferase activity towards bilirubin and 2-aminophenol in human liver. Biochem J 1979;184:705-7 . Seminal in development. 124. Strassburg CP, Manns MP, Tukey RH. Differential down-regulation of the UDP-glucuronosyltransferase 1A locus is an early event in human liver and biliary cancer. Cancer Res 1997;57:2979-85 125. Tiribelli C, Ostrow JD. New Concepts in Bilirubin and Jaundice: report of the Third International Bilirubin Workshop, April 6-8, 1995, Trieste, Italy. Hepatology 1996;24:1926-311 126. Zaya M, Hines R, Stevens J. Epirubicin glucuronidation and UGT2B7 developmental expression. Drug Metab Dispos 2006;34:2097-101 127. Collier A, Yamauchi Y, Sato B, et al. UDP-glucuronosyltransferase 1a enzymes are present and active in the mouse blastocyst. Drug Metab Dispos 2014;42:1921-5 . Earliest identification of UGt in mammalian development. 128. Burchell B, Coughtrie M, Jackson M, et al. Development of human liver UDP-glucuronosyltransferases. Dev Pharmacol Ther 1989;13:70-7 129. Burchell B, Dutton GJ. Delayed induction by phenobarbital of UDP-glucuronyltransferase activity towards bilirubin in fetal liver. Biol Neonate 1975;26:122-8 130. Hume R, Burchell A, Allan BB, et al. The ontogeny of key endoplasmic reticulum proteins in human embryonic


microsomes. Mol Pharmacol 1988;34:729-35 Seminal in UGT development.

and fetal red blood cells. Blood 1996;87:762-70 131.


132.

Cappiello M, Giuliani L, Rane A, et al. Uridine 5’-diphosphoglucuronic acid (UDPGLcUA) in the human fetal liver, kidney and placenta. Eur J Drug Metab Pharmacokinet 2000;25:161-3 Coughtrie M, Burchell B, Leakey JE, et al. The inadequacy of perinatal glucuronidation: immunoblot analysis of the developmental expression of individual UDP-glucuronosyltransferase isoenzymes in rat and human liver

.

133.

Freeze H. Congenital disorders of glycosylation and the pediatric liver. Semin Liver Dis 2001;21:501-15

134.

Barone R, Sturiale L, Palmigiano A, et al. Glycomics of pediatric and adulthood diseases of the central nervous system. J Proteomics 2012;75:5123-39

135.

Guillemette C. Pharmacogenomics of human UDP-glucuronosyltransferase


enzymes. Pharmacogenomics J 2003;3:136-58

Affiliation

Zoe Riches & Abby C Collier† † Author for correspondence University of British Columbia, Faculty of Pharmaceutical Sciences, 2405 Wesbrook Mall, Vancouver, BC V6T 1Z3, Canada Tel: +1 604 827 2380; E-mail: [email protected]

965

Regulation of uridine diphosphate-glucuronosyltransferase 1A3 activity by protein phosphorylation.

Revisiting the Latency of Uridine Diphosphate-Glucuronosyltransferases (UGTs)-How Does the Endoplasmic Reticulum Membrane Influence Their Function?

Enzymatic Redox Cascade for One-Pot Synthesis of Uridine 5'-Diphosphate Xylose from Uridine 5'-Diphosphate Glucose.

Reduced hepatic bilirubin uridine diphosphate glucuronyl transferase and uridine diphosphate glucose dehydrogenase activity in the human fetus.

Estimates of uridine diphosphate glucose in human erythrocytes.

Half-sites oxidation of bovine liver uridine diphosphate glucose dehydrogenase.

Uridine diphosphate-sugar metabolism by sycamore (Acer pseudoplatanus) cambial tissue.

Uridine diphosphate xylosyltransferase activity in cartilage from manganese-deficient chicks.

Structure of uridine diphosphate N-acetylglucosamine pyrophosphorylase from Entamoeba histolytica.

Interaction of uridine diphosphate glucose with calf liver uridine diphosphate glucose dehydrogenase. Significance of hydroxyl groups at C-3, C-4 and C-6 of hexosyl residue.

Posttranscriptional regulation of myelin protein gene expression.

Regulation of vascular function on posttranscriptional level.

Posttranscriptional regulation of oral bacterial adaptive responses.

Influence of para substituent of phenol on phospholipid dependence of uridine diphosphate-glucuronyltransferase.

Evidence for specific transport of uridine diphosphate galactose across the Golgi membrane of rat mammary gland.

Assay of glucoprotein formation from uridine diphosphate glucose by a rapid filter-paper technique.

Structures of Bacteroides fragilis uridine 5'-diphosphate-N-acetylglucosamine (UDP-GlcNAc) acyltransferase (BfLpxA).

Stimulation of steroid glucuronidation by uridine diphosphate N-acetylglucosamine and by microsomal disruption.

Cellular localization of uridine 5'-diphosphate-N-acetyl-D-glucosamine oxidoreductase activity in Citrobacter freundii.

Postnatal development of uridine diphosphate glucuronyltransferase activity towards bilirubin and 2-aminophenol in human liver.

Biosynthesis of uridine diphosphate N-acetylmannosaminuronic acid in rff mutants of Salmonella tryphimurium.

Chemical transformation of sugar nucleotides: acetylation of uridine 5'-(alpha-D-glucopyranosyl diphosphate).

Dimerization of human uridine diphosphate glucuronosyltransferase allozymes 1A1 and 1A9 alters their quercetin glucuronidation activities.

Altered sexual differentiation of hepatic uridine diphosphate glucuronyltransferase by neonatal hormone treatment in rats.