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Molecular Biology, Regulation, and Biochemistry of UDP-Glucuronosyltransferase Isozymes

Ida S. Owens, PhD, Head, Section on Genetic Disorders of Drug Metabolism
Rajat Banerjee, PhD, Visiting Fellow
Nikhil K. Basu, PhD, Staff Scientist
Mousumi Basu, BS, Technician Training Fellow
Kushal Chakraborty, PhD, Visiting Fellow
Sunit K. Chakraborty, PhD, Visiting Fellow

UDP-glucuronosyltransferase (UGT) isozymes, distributed primarily in liver, kidney, and gastrointestinal tract, carry out the essential function of converting innumerable and frequently encountered, but structurally diverse, lipophilic chemicals to glucuronides. Such lipophiles include toxic metabolites, dietary constituents, environmental agents/carcinogens, and therapeutics. Conversion of chemicals to glucuronides inactivates them and hastens their excretion, which prevents tissue accumulation and toxicities. Neurotoxic bilirubin is the most important endogenous substrate, followed by genotoxic catechol estrogens. Given that the UGT isozyme system prevents bilirubin neurotoxicities in children, inactivates common environmental mutagens and carcinogens, and converts many therapeutics prematurely, it is important to understand the mechanism of glucuronidation. Such an understanding would allow development of methods and strategies for accelerating the removal of toxic chemicals, while extending the therapeutic benefits of glucuronidatable medications. Moreover, greater knowledge of the enzymatic mechanism(s) and properties that enable a limited number of endoplasmic reticulum–bound UGTs to convert numerous structurally diverse lipophiles would enhance our ability to prevent chemical-derived diseases. Our cloning and characterization of the bilirubin-specific UGT1A1 enabled us to identify the genetic defect in hyperbilirubinemic Crigler-Najjar children. Additionally, we discovered and characterized the novel complex locus UGT1, which encodes UGT1A1 and 12 other UGTs that are independently regulated but share a common carboxyl terminus. We also cloned and characterized the catechol estrogen–metabolizing UGT2B7 and the dihydrotestosterone-metabolizing UGT2B15. UGT2B7 is important for inactivating genotoxic catechol estrogens associated with initiation of breast cancer; inactivation of dihydrotestosterone by UGT2B15 reduces the level of the critical androgen that is associated with prostate cancer.

Expansion of the phosphorylation requirement of UGT1 family members

As noted, the special properties and enzymatic mechanism(s) that enable endoplasmic reticulum (ER)–bound UGT isozymes to convert innumerable structurally diverse lipophiles to excretable glucuronides remain unknown. Inhibition of cellular UGT1A7 and UGT1A10 activities and of time- and concentration-dependent [³³P]orthophosphate incorporation into these immunoprecipitable proteins following exposure to curcumin or calphostin-C indicated that the isozymes are phosphorylated (Basu et al., Proc Natl Acad Sci USA 2005;102:6285). Furthermore, inhibition of UGT phosphorylation and activity by treatment with PKCε–specific inhibitor peptide supported the conclusion that PKC is involved. Co-immunoprecipitation, co-localization via immunofluorescence, and cross-linking studies of PKCε and UGT1A7His revealed that the proteins reside within 11.4 Å of each other. Moreover, mutation of three PKC sites in each UGT isozyme demonstrated that T73A/G and T202A/G caused null activity, whereas S432G–UGT1A7 caused a major shift of the isoenzyme’s pH optimum from 8.5 to 6.4 as well as changes in substrate specificity to include 17β-estradiol. S432G-UGT1A10 exhibited a minor pH shift without changing substrate specificity. We confirmed PKCε involvement by demonstrating, first, that PKCε overexpression enhanced the activity of UGT1A7 but not that of its S432 mutant and, second, that S432G-UGT1A7 but not UGT1A7 glucuronidates 17β-[¹⁴C]estradiol. Consistent with these observations, treatment of UGT1A7–transfected cells with PKCε–specific inhibitor peptide or general PKC inhibitors increased 17β-estradiol catalysis between 5- and 11-fold, with parallel decreases in the level of phospho-serine-432. This is a novel mechanism involving PCC–mediated phosphorylation of UGT such that phospho-serine/threonine regulates substrate specificity in response to chemical exposures, possibly conferring survival benefit.

Dependence of UGT2B7 activity on phosphorylation by c-Src kinase

We discovered that all UGTs examined require phopshorylation in order to carry out glucuronidation. Whereas the requirement differs for each UGT, the UGT2B7 isozyme, which preferentially metabolizes genotoxic catechol estrogens associated with breast cancer initiation, undergoes required tyrosine phosphorylation at two different residues. UGT2B7 incorporation of immunoprecipitable [³³P]orthophosphate following expression in COS-1 confirmed phosphorylation. The activity of Y236F-UGT2B7 and Y438F-UGT2B7 mutants was reduced by 90–100%, indicating that tyrosine phosphorylation is required. Partial inhibition of activity by Src-specific PP2 with loss of anti–phospho-Y438-2B7, combined with similar disruption of co-localization and cross-linking of active c-Src and UGT2B7, strongly indicated that Src phosphorylates UGT2B7. Affinity-purified UGT2B7His, which was initially expressed in SYF^−/− cells and phosphorylated in vitro with Src and [³³P]ATP versus [³³P]ATP alone, confirmed that Src supports UGT2B7 phosphorylation based on both markedly enhanced anti–phospho-Y438-2B7 sites and activity. Additionally, AKAP12-siRNA treatment destroyed 70−100% of this ER–specific and c-Src–binding scaffold, active Src, phospho–Y438-UGT2B7 and UGT2B7 activity, but not the level of the UGT2B7 protein. Moreover, we found concordant dramatic losses of active c-Src, c-Src activity, UGT2B7, phospho-Y438-UGT2B7, and UGT2B7 activity in breast carcinomas compared with matching controls, indicating that c-Src supports functional UGT2B7. Finally, UGT2B7-transfection of COS-1 cells provided greater than 80% protection against 4-OH-estrone−based depurination. While c-Src supports the required and regulated phosphorylation of UGT2B7, which is disrupted in breast-carcinomas, functional UGT2B7 protects against catechol estrogen depurination that is linked to tumor initiation.

Dependence of UGT2B15 on PKC phosphorylation but independent downregulation by Src kinase phosphorylation

Glucuronidation of dihydrotestosterone (DHT) by UGT2B15 or UGT2B17 is the primary reaction for inactivating this potent androgen, while cytochrome P450 also converts the hormone to 5-α-androstane-3α,17β-diol, a metabolite that is more efficiently glucuronidated. The finding that these UGT2B isozymes are distributed primarily in prostate and testis, as well as liver, suggests that glucuronidation plays an important role in the maintenance of DHT levels in hormone-responsive tissues. Consistent with our previous findings, transient down-regulation of UGTs following curcumin exposure signified that the isozyme required regulated phosphorylation. Curcumin transiently down-regulated UGT2B15, while the typical PKC inhibitors calphostin-C, BIM, Gö6976, and Rötlerin irreversibly inhibited activity by 80–100%. In addition, the c-Src–specific PP2 inhibitor and c-Src-siRNA inhibited activity in a concentration-dependent manner. Computer-detected PKC and tyrosine kinase (TK) sites demonstrated that the S172A and S124A mutants were 100% and 90% inactivated, respectively, while the Y237F and Y99F were 85% and 50% inactivated, respectively. In addition the JNK–specific inhibitor SB230580 caused modest inhibition while the P³⁸MAP kinase–specific inhibitor DATS caused modest activation/inhibition of UGT2B15 activity, which indicated that c-Src plays a secondary role in phosphorylation. With an anti-His preparation, UGT2B15His and PKCα co-immunoprecipitated, but the proteins were dissociated by pretreatment with BIM, Gö6976, or Rötlerin. Our findings indicate that UGT2B15 requires PKCα phosphorylation at S172, but that c-Src likely participates in a supportive or regulatory role at Y237.

Dependence of UGT2B17 on PKC phosphorylation

Because UGT2B17 exhibits the most robust glucuronidation of dihydrotestosterone (DHT) under in vitro condictions, it is predicted the isozyme catalyzes the primary inactivating reaction of the androgen under in vivo conditions. Similar to its effects on UGT2B15, we found that curcumin transiently down-regulates UGT2B17, which suggested that the isozyme also requires phosphorylation. Although UGT2B17 has two PKC and two tyrosine kinase sites, the enzyme is inhibited in a concentration-dependent manner as follows: 80% by calphostin-C, 50% by BIM, 60% by Rotlerin, and 50% by Gö6976. UGT2B17 is activated by c-Src–specific inhibitor PP2 (50%) and c-Src–siRNA (20%), with minor effects of the c-Src scaffold protein AKAP12-siRNA. Following mutations at the PKC and tyrosine kinase sites S422A, Y99F, Y237F, and S172F, inhibition ranged from 40–100%. Given that all mutants except Y237F and S172A were activated by PP2 treatment, Y237 is likely to be the site of c-Src suppression. In conclusion, our results indicate that UGT2B17 requires PKC phosphorylation at S172 while c-Src appears to suppress UGT2B17 activity via a regulatory mechanism likely located at Y237.

Regulation of UGT phosphorylation via signaling

Our finding of rapid, reversible downregulation of human UGT in LS180 cells following curcumin-treatment led to the discovery that UGTs require phosphorylation. Our aim was to determine relevant kinases and mechanism(s) regulating phosphorylation of constitutive UGTs in LS180 cells and 10 different human UGTcDNA–transfected COS-1 systems. Time- and concentration-dependent inhibition of immunodetectable [³³P]orthophosphate in UGTs and PKCε following treatment of LS180 cells with curcumin or the PKC inhibitor calphostin-C suggested that UGT phosphorylation is supported by active PKC(s). Immunofluorescence and co-immunoprecipitation studies with UGT–transfected cells showed co-localization of UGT1A7His with PKCε and of UGT1A10His with PKCα or PKCδ; the co-localization was disrupted by PKC inhibitors. Inhibition of UGT activity by PKCε–specific antagonist peptide or by PKCε–targeted destruction with PKCε–specific siRNA and activation of curcumin-downregulated UGTs with typical PKC agonists verified a central role of PKC in glucuronidation. Moreover, in vitro phosphorylation of nascent UGT1A7His by PKCε confirms that it is a bona fide PKC substrate. Finally, catalase or herbimycin-A inhibition of constitutive or hydrogen peroxide–activated UGTs implicated ROS-related oxidants as second messengers in maintaining constitutive PKC–dependent signaling—evidently sustaining UGT phosphorylation and activity. As cells use signal transduction collectively to detect and respond appropriately to environmental changes, these findings, combined with our earlier demonstration that specific phospho-groups in UGT1A7 determined substrate selections, suggest that regulated phosphorylation allows adaptations regarding differential phosphate utilization by UGTs for the isozymes to function efficiently.

Tertiary structure of UGT(s)

To obtain further details of the requirements of phosphorylation of ER-bound UGTs, we attempted to purify a catalytically active UGT protein for structural analysis. UGT1A7- and UGT2B7-cDNAs, adapted with thrombin/his/myc affinity ligands, not only establish a highly effective UGT-solubilizing system that retains activity but also permit us to isolate UGT-containing complexes involved in PKC and/or tyrosine kinase signaling pathways similar to those described for other cellular processes. The complex contained one 58 kDa UGT1A7His and one 110 kDa β-COP to two 29 kDa 14-3-3 phospho-serine chaperone proteins. For PKC-dependent signaling, we found that UGT1A7 associated with RACKε in a 225 kDa adapter complex that included the phosphoserine-dependent 14-3-3 protein. Mutation of UGT1A7His at its 14-3-3 binding sites led to marked lability of solubilized UGT1A7His. Like all UGTs, UGT1A7 has two 14-3-3 binding sites: S162 and T403. Mutations at these sites indicate that 14-3-3 also stabilizes UGT stored at 4°C. These important findings should enable us to isolate a UGT and carry out analysis of its tertiary structure as well as identify events and components involved in phosphorylation-dependent signaling. It is notable that 14-3-3 binding sites exist in all UGTs. We confirmed binding of UGTs to phosphoserine–14-3-3 by co-immunoprecipitation and/or co-localization with UGT1A1His, UGT1A6His, UGT1A7His, UGT1A10, or UGT2B7His. In summary, our studies indicate that UGT1A7 exists as a cellular complex(es) that sustains activity via protein kinase(s) signaling. Our findings lay the foundation for further studies concerning this critical endogenous chemical defense enzyme system.

Structural analysis and identification of the common donor-substrate binding site in UGT1A10

The critical role that the UGT isozyme system plays in protecting the body against endogenous and exogenous chemicals is well known. Each isozyme links glucuronic acid donated by UDP-glucuronic acid to a lipophilic acceptor substrate to generate a glucuronidated metabolite. Because of the difficulties associated with purifying ER–bound UGTs for structural studies, we carried out homology-based computer modeling to aid analysis. The search found structural homology in Escherichia coli UDP-galactose 4-epimerase. Consistent with predicted similarities involving the common UDP-moiety in substrates, Lineweaver-Burke plots showed that UDP-glucose and UDP-hexanol caused competitive inhibition. Among the predicted binding sites N292, K314, K315, and K404 in UGT1A10, there were two informative sets of mutants: the K314R/Q/A/E/G mutants had null activities; that of K404R was 2.7-fold higher; and K314/E had 50% less activity. Scatchard analysis of binding of the affinity ligand 5-azido-uridine-[β-³²P]-diphosphoglucuronic acid to purified UGT1A10-His or UGT1A7-His revealed high- and low-affinity binding sites. 2-Nitro 5-thiocyanobenzoic acid (NTCB)–digested UGT1A10-His bound with radiolabeled affinity ligand revealed an 11.3-kDa and a 14.3-kDa peptide associated with K314 and K404, respectively. Similar treatment of UGT1A10His-K314A bound with the ligand lacked both peptides; UGT1A10-HisK404R and UGT1A10-HisK404E showed 1.3-fold greater and 50% less label in the 14.3-kDa peptide, respectively, compared with UGT1A10-His, without affecting the 11.3-kDa peptide. Scatchard analysis of binding data of the affinity ligand to UGT1A10His-K404R and UGT1A10His-K404E showed a 6-fold reduction and a large increase in Kd, respectively. Our results indicate that K314 and K404 are required UDP-glcA binding sites in UGT1A10, that K404 controls activity and high affinity sites, and that K314 and K404 are strictly conserved in 70 aligned UGTs, except for S321—equivalent to K314—in UGT2B15 and UGT2B17 and I321 in the inactive UGT8, which suggests that UGT2B15 and UGT2B17 contain suboptimal activity. Hence our data strongly support UDPglcA binding to K314 and K404 in UGT1A10.

Publications

Basu NK, Kole L, Basu M, Chakraborty K, Mitra PS, Owens IS. The major chemical detoxifying system of UDPglucuronosyltransferases requires regulated phosphorylation supported by protein kinase C. J Biol Chem 2008 283:23048-23061.
Banerjee R, Pennington MW, Garza A, Owens IS. Mapping the UDP glucuronic acid binding site in UDP-glucuronosyltransferase-1A10 by homology-based modeling: confirmation with biochemical evidence. Biochemistry 2008 47:7385-7392.
Mitra PS, Basu NK, Owens IS. Src supports UDP-glucuronosyltransferase-2B7 detoxification of catechol estrogens associated with breast cancer. Biochem Biophys Res Commun 2009 382:651-656.
Basu NK, Kole L, Basu M, McDonagh AF, Owens IS. Targeted inhibition of glucuronidation markedly improves drug efficacy in mice—a model. Biochem Biophys Res Commun 2007 360:7-13.

Collaborators

Antony McDonagh, PhD, University of California San Francisco, San Francisco, CA
Masahiko Negishi, PhD, Laboratory of Reproductive and Developmental Toxicology, NIEHS, Research Triangle Park, NC
Juan Rivera, PhD, Molecular Immunology and Inflammation Branch, NIAMS, Bethesda, MD

Contact

For further information, contact ida.owens2@nih.gov.

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