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

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

UDP-glucuronosyltransferase (UGT) isozymes, distributed primarily in liver, kidney, and the gastrointestinal tract, carry out the essential role of converting to glucuronides innumerable, frequently encountered, structurally diverse lipophilic chemicals, including toxic metabolites, dietary constituents, environmental carcinogens, and therapeutics, whose conversion hastens excretion and prevents their accumulation in tissue and their toxic effects in the body. Neurotoxic bilirubin is the most important endogenous substrate. Given that the UGT isozymes prevent bilirubin neurotoxicities in children and inactivate common mutagens and carcinogens but prematurely clear therapeutic chemicals, it is important to understand the mechanism of glucuronidation in order to develop methods and therapeutics for accelerating the removal of toxic chemicals while retaining medications to achieve maximal therapeutic benefits. Moreover, the enzymatic mechanism(s) and properties that enable a limited number of endoplasmic reticulum–bound UGTs to convert numerous structurally diverse lipophiles to innocuous glucuronides remain unknown. We discovered and characterized UGT1A, the novel complex locus encoding 13 UGT genes, that is organized to share a common carboxyl terminus. We also cloned UGT2B7 and UGT2B15 and characterized catalysis. Thus, an important research goal is to determine the properties, mechanism(s), and molecular events controlling these isozymes in order to understand how exogenous as well as endogenous chemicals lead to the disease process.

Control of the UGT1 family of isozymes by PKC phosphorylation

Basu N, Banerjee, Garza1; in collaboration with Rivera

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 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 PKC involvement. 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-1A7 but not UGT 1A7 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. We report a novel mechanism involving protein kinase C–mediated phosphorylation of UGT such that phospho-serine/threonine regulates substrate specificity in response to chemical exposures, possibly conferring survival benefit.

Expansion of the phosphorylation requirement of UGT1 family members

Basu N, Mbas-Jones,² Chang³

Inhibition by curcumin or calphostin-C of recombinant UGT1A6 and UGT1A9 expressed in COS-1 cells provides further evidence that each UGT isozyme requires phosphorylation. Time-dependent inhibition of UGT1A6 by curcumin showed a modest reversal by 5 hours. The equivalent mutation, S434G/A/D/R/K, in UGT1A6 caused a shift from a single broad pH optimum to two pH optima. For UGT1A9, the equivalent mutation, S432G/A/D/E, caused progressive diminution of activity for mycophenolic acid. Similarly, mutations inserted into the UGT1A9-His construct resulted in reduced activity as assessed by Western blot. Curcumin and calphostin-C inhibited UGT1A9 activity in a dose-dependent manner. Whereas reversal of 95 percent inhibition by curcumin occurred in 5 hours for UGT1A9 as a consequence of the residual 5 percent metabolism, reversal was much slower for UGT1A6, which did not metabolize the agent. The evidence indicates that phospho-serine/threonine in UGT1A6 and UGT1A9 directly or indirectly controls activity. Thus, our cumulative evidence indicates that each UGT requires phosphorylation.

Dependence of UGT2B7 activity on phosphorylation by c-Src kinase

Chakraborty K, Basu N, Mitra,⁴ Garza¹

Earlier, we cloned UGT2B7 and demonstrated that it metabolizes genotoxic catechol estrogens produced by the mammary gland and other estrogen target tissues and that are associated with initiation of breast cancer. UGT2B7 is the putative relevant isozyme that detoxifies these endogenous estrogen metabolites in the mammary gland. For UGT2B7, tyrosine kinase (TK) phosphorylation sites, the enzyme’s incorporation of immunoprecipitable [³³P]orthophosphate, and inhibition by TK inhibitors are all consistent with tyrosine phosphorylation, a claim further supported by the following observations: mutational studies have shown that phosphorylation of only Y236 and Y438 is required; PP2 [(4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine)], a potent and selective inhibitor of the Src-family tyrosine kinase, specifically inhibits Src with loss of phospho-Y438-UGT2B7 content; and overexpression of Src/active Src, but not of dominant-negative Src, significantly increases UGT2B7 activity and the level of phospho-Y438. PP2 disrupts co-localization of Src/active Src and UGT2B7, and UGT2B7 and Src/active Src can be cross-linked, demonstrating that UGT2B7 and Src/active Src interact in situ. AKAP12-siRNA–dependent depletion of SrcTK organization, involving the ER-scaffold protein AKAP12, in UGT2B7-transfected COS-1 cells caused 70 percent destruction of AKAP12, 80 percent loss of UGT2B7 activity, and 90 percent loss of phospho-Y438-UGT2B7 without affecting the UGT2B7 protein level. This evidence supports an AKAP12-dependent signaling network. Expression of UGT2B7 in SYF^+/− or SYF^+/+ fibroblast cells led to 2- to 4-fold higher activity than that in SYF^−/− cells. In vitro treatment of solubilized UGT2B7-containing microsomes from SYF^−/− cells with SrcTK and [γ³³P]ATP caused the microsomes to incorporate 12 times more label with markedly higher phospho-Y438-UGT2B7 content than after [γ³³P]ATP treatment alone; similarly microsomes treated with unlabeled ATP showed a 5-fold increase in UGT2B7 activity. Importantly, dramatic losses of Src activity and Src/active Src content, which paralleled loss of UGT2B7 activity and phospho-Y438-UGT2B7 in breast carcinomas compared with control tissues, are consistent with Src phosphorylation of UGT2B7. Src phosphorylation of UGT2B7, which detoxifies genotoxic catechol estrogens associated with DNA depurination and tumor initiation, indicates that UGT2B7 effectively behaves as an Src-dependent tumor suppressor.

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

Chakraborty K, Banerjee, Basu N

Earlier, we cloned prostate- and testis-distributed UGT2B15 and demonstrated that it metabolizes several androgen intermediates as well as dihydrotestosterone, which, at elevated concentrations, is associated with benign prostate hyperplasia and prostate cancer. Treatment of UGT2B15-transfected COS-1 cells revealed that both PKC- and tyrosine kinase inhibitors downregulated activity. Appropriate concentrations of the PKC-specific inhibitor calphostin-C inhibited activity by 97 percent. Computer analysis showed that UGT2B15 contains three PKC and two tyrosine phosphorylation sites. Mutation of phosphorylation sites in UGT2B15 demonstrated that a single PKC site is required for activity. Unlike trivial levels of catechol-estrogen–metabolizing UGT2B7 activity in transfected SYF^−/− cells, UGT2B15 expressed its highest activity in SYF^−/− cells; such activity was inhibited by 64 and 88 percent when expressed in SYF^+/− and SYF^+/+ cells, respectively. As a broad screen to determine other pathways involved in controlling UGT2B15 activity, we carried out rescue experiments following expression in COS-1 cells. The Src-specific inhibitor PP2, the p38MAP kinase–specific inhibitor SB230580, and the JNK kinase–specific inhibitor DATS (diallyl trisulfide) increased activity between 2- and 4-fold, indicating that the p38MAP kinase and JNK kinase pathways are likely upstream of Src kinase, which downregulates UGT2B15. Our evidence indicates that c-Src inhibits UGT2B15.

Dependence of UGT2B17 on PKC phosphorylation

Park, Basu N

Like UGT2B15, prostate- and testis-distributed UGT2B17 metabolizes several androgen-intermediates and dihydrotestosterone, but its activity is 5-fold higher than UGT2B15. Thus, UGT2B17 is also associated with benign prostate hyperplasia and prostate cancer and is likely as important as UGT2B15. The two proteins are differentially distributed in luminal (UGT2B15) and basal (UGT2B17) cells. Using UGT2B17-transfected COS-1 cells, we demonstrated that PKC inhibitors downregulated activity; appropriate concentrations of the PKC-specific inhibitor calphostin-C inhibited PKC activity by 97 percent. Computer analysis revealed that UGT2B17 contains two PKC- and two tyrosine-phosphorylation sites. Mutation analysis of UGT2B17 phosphorylation sites demonstrated that, parallel to UGT2B15, only S172 alterations caused null activity, whereas S422A, Y99F, and Y237F lost, respectively, 20, 66, and 75 percent activity. Moreover, UGT2B17, like UGT2B15, has higher activity in SYF^−/− cells than when expressed in COS-1 cells. Currently, we are further characterizing UGT2B17.

Regulation of UGT phosphorylation via signaling

Basu N, Kole,⁵ Garza¹ Mitra,⁴

Catalase or herbimycin-A inhibition of constitutive or hydrogen peroxide–activated UGTs demonstrated that ROS-related oxidants behave as second messengers in maintaining constitutive PKC-dependent signaling, evidently sustaining UGT phosphorylation and activity. Given that cells use signal transduction collectively to detect and respond appropriately to a changing environment, our study, combined with our earlier demonstration of phospho-dependent substrate selections by UGT, suggests that, to function efficiently, regulated phosphorylation allows adaptations via differential phosphate utilization by UGTs.

Basu NK, Kole L, Basu M, Chakraborty K, Mitra PS, Owens IS. The major chemical detoxifying system of UDP-glucuronosyltransferases requires regulated phosphorylation supported by protein kinase C. J Biol Chem 2008;283:23048-23061.

Tertiary structure of UGT(s)

Garza¹ Basu N, Basu M

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 TK 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 includes the phosphoserine-dependent 14-3-3 protein. Mutation of UGT1A7His at its 14-3-3 binding sites led to marked lability of solubilized 1A7His. 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 surrounding phosphorylation-required 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 UGT1A1 His, 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

Banerjee, Pennington⁶

Given that UGT isozymes generate inactive chemical glucuronides by linking glucuronic acid donated by UDP-glucuronic acid to lipophilic acceptor substrates, thereby enhancing their excretion, it is important to determine the binding properties and sites for the donor substrate. It would be difficult to purify ER-bound UGTs for structural studies, so we carried out homology-based computer modeling to aid analysis. Consistent with predicted similarities involving the common UDP moiety in substrates UDP-glucose and UDP-glucuronic acid, the model identified structural homology in Escherichia coli UDP-galactose 4-epimerase and UGT1A10; the model predicted binding sites at N292, K314, K315, and K404 in 1A10. Two informative sets of UGT1A10 mutants, K314R/Q/A/E /G and K404R/E, had null activities and 2.7-fold higher activity, respectively. Scatchard analysis of the binding of the affinity ligand 5-azido-uridine-[β-32P]-diphosphoglucuronic acid to UGT1A10-His or UGT1A7-His revealed high- and low-affinity binding sites. Discontinuous SDS-PAGE of 2-nitro-5-thiocyanobenzoic acid–digested UGT1A10-His bound to radiolabeled affinity ligand revealed 11.3 and 14.3 kDa peptides associated with K314 and K404, respectively, with both peptides undetectable in UGT1A10-K314 mutants. Increased basicity of residue 404 led to increased 14.3 kDa peptide content and high-affinity binding sites and Kd, accounting for increased UGT activity. Notably, K314 and K404 are strictly conserved in 70 aligned UGTs, except for S321—which is equivalent to K314—in UGT2B15 and UGT2B17 and for I321 in the inactive UGT8, suggesting that UGT2B15 and UGT2B17 are suboptimally active. Hence, our data strongly support UDPglcA binding to K314 and K404 in UGT1A10.

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.

Marked improvement in drug efficacy by targeted inhibition of glucuronidation in mice

Basu N, Kole,⁵ Basu M; in collaboration with McDonagh

The finding that UGTs require protein kinase C–mediated phosphorylation is important information that allows manipulation of this critical glucuronidating system. Given that UGTs may be inactivated by downregulating PKCs with reversibly acting dietary curcumin, we determined the impact of gastrointestinal (GI) glucuronidation on free drug uptake and efficacy by using the immunosuppressant mycophenolic acid (MPA) in mice. Expressed in COS-1 cells, mouse GI-distributed Ugt1a1 glucuronidates curcumin and MPA and undergoes irreversible and reversible dephosphorylation by the PKC-specific inhibitor calphostin-C and the general kinase inhibitor curcumin, respectively, with parallel effects on activity. Moreover, oral administration of curcumin to mice reversibly inhibits glucuronidation in GI tissues. Finally, successive oral administration of curcumin and MPA to antigen-treated mice raises free MPA in serum and causes 9-fold immunosuppression. Using MPA as a model, our results indicate that targeted inhibition of GI glucuronidation in mice markedly improved free chemical uptake and efficacy.

Basu NK, Kole L, Basu M, McDonagh AF, Owens IS. Targeted inhibition of glucuronidation markedly improves drug efficacy in mice—a model. Biochem Biophy Res Commun 2007;360:7-13.

¹ Amanda Garza, BS, former Predoctoral Fellow
² Chimere Mbas-Jones, former Summer Student
³ Elaine Chang, former Summer Student
⁴ Partha S. Mitra, PhD, former Visiting Fellow
⁵ Labanyamoy Kole, PhD, former Postdoctoral Fellow
⁶ Matthew Pennington, former Summer Student

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

For further information, contact owens@helix.nih.gov.