Ida S. Owens, PhD, Head, Section on Genetic Disorders of Drug Metabolism
Nikhil K. Basu, PhD, Research Fellow
Rajat Banerjee, PhD, Visiting Fellow
Sunit K. Chakraborty, PhD, Visiting Fellow
Partha S. Mitra, PhD, Visiting Fellow

UDP-glucuronosyltransferase (UGT) isozymes carry out the essential function of transforming innumerable, frequently encountered, structurally diverse lipophilic chemicals, including toxic metabolites, dietary constituents, environmental carcinogens, and therapeutics to glucuronides, which hastens excretion, preventing tissue accumulation and toxic effects in the body. Neurotoxic bilirubin is the most important endogenous substrate. Given that the isozymes prevent bilirubin neurotoxicities in children, inactivate common mutagens and carcinogens, and prematurely clear therapeutic chemicals, it is important to understand the mechanism of glucuronidation in order to hasten the removal of toxic chemicals while retaining medications so that they can achieve their maximal therapeutic benefits. Moreover, researchers have yet to identify the enzymatic mechanism(s) and properties that enable a limited number of endoplasmic reticulum-bound UGTs to convert numerous structurally diverse lipophiles into innocuous glucuronides. We have, however, uncovered and characterized the novel complex UGT1A locus, which encodes 13 UGT genes organized to share a common carboxyl terminus, and have cloned UGT2B7 and UGT2B15 and characterized catalysis. Therefore, our aim is to understand the properties and mechanism(s) of these various isozymes that enable us to detoxify a vast number of agents in order to maintain chemical homeostasis.

Phosphorylation of UGT1 family members by PKC with signaling

Basu, Garza, ¹ Mitra, Banerjee, Rivera

Even though UGT isozymes conjugate to glucuronic acid several chemical toxins present in our daily diet and environment, the 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 [33P]orthophosphate incorporation into immunoprecipitable proteins following exposure to curcumin or calphostin-C indicates that the isozymes are phosphorylated. Furthermore, inhibition of UGT phosphorylation and activity by treatment with PKCepsilon-specific inhibitor peptide supports PKC involvement. Computer analysis revealed that each UGT isozyme has four to six protein kinase C (PKC) sites. Co-immunoprecipitation, co-localization using immunofluorescence, and cross-linking studies of PKCepsilon and UGT1A7His revealed that the proteins reside within 11.4 Å of each other. Mutation of three PKC sites in each UGT isozyme demonstrated that T73A/G and T202A/G caused null activity, whereas S432G-UGT1A7 caused major shifts in the enzyme's pH optimum from 8.5 to 6.4 and in substrate specificities. We confirmed PKCepsilon involvement by demonstrating that (1) PKCepsilon overexpression enhances UGT1A7 activity but not that of its S432 mutant and (2) S432G-1A7 but not UGT1A7 converts 17β [14C]estradiol. Consistent with these observations, treatment of 1A7-transfected cells with PKCepsilon-specific inhibitor peptide or general PKC inhibitors dramatically increased 17β-estradiol catalysis with parallel decreases in phosphoserine-432. Thus, protein kinase C-mediated phosphorylation of serine/threonine in UGT regulates substrate specificity, which possibly confers survival benefit.

Expansion of the phosphorylation requirement of UGT1 family members

Mbas-Jonas, ² Change, ³ Basu

Inhibition of recombinant UGT1A6 and UGT1A9, expressed in COS-1 cells, by curcumin or calphostin-C is further evidence that each UGT isozyme requires phosphorylation. Time-dependent inhibition of UGT1A6 by curcumin showed a modest level of reversal by five hours. While mutants at T73/T74 or T202/T206 in each isozyme abolished activity, S432G/S434G mutants for UGT1A7 and 1A10 were not affected, exhibited a minor shift in pH optimum or a marked shift in pH optimum from 8.5 to 6.4. S434G/A/D/R/K, the equivalent mutant in UGT1A6, showed a shift from a single broad pH optimum to two pH optima. S432G/A/D/E, the equivalent mutant of UGT1A9, showed a progressive diminution of activity for mycophenolic acid. Similarly, mutants inserted into the UGT1A9-His construct expressed reduced activity. Curcumin and calphostin-C inhibited UGT1A9 activity in a dose-dependent manner. Whereas reversal of 95 percent inhibition by curcumin occurred within five hours for UGT1A9, reversal was much slower for UGT1A6, which does not metabolize curcumin. The evidence indicates that phosphoserine/threonine in 1A6 and 1A9 directly or indirectly controls activity. Our cumulative evidence indicates that each UGT requires phosphorylation.

Purification of UGT isozymes

Garza, ¹ Basu

To characterize the phosphorylation required for UGTs, we attempted to purify a catalytically active UGT protein for structural analysis. UGT1A7cDNA, adapted at the 3′ end to specify Thrombin/His/Myc sites at the carboxyl end and expressed by pBlueBac-based baculoviral and pSVL-based vectors in Sf9 insect cells and COS-1 cells, respectively, allows removal of the His-tag ligand site. Given that membrane-bound UGT requires an effective but non-inactivating detergent system, we developed an effective two-detergent system that allowed us to partially affinity-purify UGT1A7His and UGT1A10His. UGT1A7His eluted from the affinity chromatography column reproducibly contains 15-20 proteins. One chromatographed sample contains a 225-kDa complex of UGT1A7, β-COP, and a phosphoserine-binding protein (PBP) (1:1:2) as well as a 300 to 600-kDa complex of UGT1A7 with various PKC isozymes, select tyrosine kinase (sTK), and β-COP. We confirmed involvement of the PBP by its co-localization with UGT1A1His, UGT1A6His, UGT1A7His, UGT1A10, or UGT2B7His. In addition, each UGT contains two consensus sequences for binding to the PBP; mutation of a single PBP site in UGT1A7 caused between 50 and 90 percent loss of activity.

Select tyrosine kinase-dependent phosphorylation of UGT2B7: a marker for breast cancer

Mitra, Basu, Garza ¹

The human UDP-glucuronosyltransferase 2B7 (UGT2B7) is probably the isozyme that detoxifies genotoxic catechol-estrogens and the critical bile hyodeoxycholic acid; we have found that the protein requires phosphorylation. Consistent with the presence of tyrosine kinase (TK) and PKC phosphorylation sites, UGT2B7 is downregulated by the TK inhibitors curcumin, genistein, herbimycin, and PP2. Mutagenesis indicates that phosphorylation of Y236 and Y438 is required but not that of T123, S132, or S437. UGT2B7 incorporates immunoprecipitable [33P]orthophosphate; phosphorylation progressively decreases as phosphorylation sites are mutated. Inhibition of UGT2B7 activity and of Y438-UGT2B7 phosphorylation by the selective TK (sTK)-specific inhibitor PP2, but not by PP3, indicates that sTK phosphorylates 2B7. In addition, by using [33Pgamma]ATP, we found that sTK phosphorylates UGT2B7-derived peptides containing a tyrosine kinase site five times more effectively than mutant peptides. Moreover, enhancement of both UGT2B7 activity and its Y438-phosphorylation by co-transfection of COS-1 cells with wild-type or activated sTK, but not by dominant-negative sTK, and cross-linking of 2B7 and sTk, which was dissociated by PP2 pretreatment, indicate that sTK phosphorylates the UGT. Additional evidence that sTK phosphorylates 2B7 is derived from the following observations: (1) mutation of sTK binding sites in 2B7 caused decreases in antiUGT2B7 pull-down of sTK in solubilized transfected COS-1 cells; and (2) PP2 disrupted co-localization of active sTK and 2B7. Normal breast development requires sTK, which is also associated with breast carcinoma; we showed that UGT2B7-specific activity in eight pairs of normal/tumor breast tissue sets is closely associated with active sTK but independent of total sTK. Our findings suggest that the capacity of 2B7 to detoxify genotoxic catechol estrogens is dependent on active sTK in this estrogen-responsive tissue.

Structural analysis of UGT

Banerjee, Pennington, ⁴ Basu

Given that UDP-glucuronosyltransferases are bound to the endoplasmic reticulum and are thus difficult to purify for crystallization, structural analysis of these critical detoxifying isozymes has been difficult. In fact, all early attempts to find structurally determined proteins homologous to the UGT1 isozymes failed. However, Matthew Pennington, a summer college student, carried out computer- and homology-based molecular modeling searches of the Protein Data Bank (PDB). Using a Silicon Graphics O2® workstation and Insight II software (Molecular Simulation, Inc.) with the Homology, MODELER, Discover, Biopolymer, and SeqFold expansion modules, he searched for structural matches for UT1A10. The new secondary-structure prediction-based, sequence homology search engine SeqFold module uses a threading technique to identify potentially homologous proteins based on the predicted secondary structure of the target sequence and the known secondary structures within structurally solved protein databases restricted to a non-redundant version of the PDB. Pennington created a protein homologue by using SeqFold and then applied the structure of that sequence homologue to map the sequence of an unknown structure to a set of three-dimensional coordinates. He first investigated protein regions near the core and on well-conserved sections and then built onto the core regions of poor conservation by using a fragment database and conformation searching techniques. Pennington next optimized the initial model by using a simulated-annealing technique. Given that any sequence can be made into a model based on any protein, Pennington thoroughly evaluated the model for feasibility. After establishing a suitability model, he analyzed the model similar to that for crystallographic structure.

He used SeqFold to identify structural homologues low on direct sequence-sequence identity to the target protein. Although SeqFold uncovered many low-identity homologues that bound to substrates similar to those bound by UGT, the most prominent enzyme with the most chemically proper structure was a UDP-galactose→UDP-glucose isomerase, known as 1XEL in the PBD. Pennington located highly homologous regions of UGT1A10 and 1XEL and confirmed those involved in binding to UDP-glucose, which is clearly analogous to the donor substrate UDP-glucuronic acid (UDPGA) common to all UGTs. He identified primarily two lysines (positions 314 and 315) and one asparagine (position 292) predicted to be critical in identifying the uracil-diphosphate portion of UDPGA, the donor substrate. Our recent studies show that mutants at 314, but not at 315, of UGT1A10 caused null activity. Activity for mutants at lysine 404, shown to be proximal to lysines 314 and 315 in the predicted UDPGA binding site, was not affected and did not have a sharper pH optimum.

Clinical relevance of controlling glucuronidation

Basu, Kole, ⁵ McDonagh

We are exploiting our characterization of the phosphorylation requirement of human UGT isozymes to determine whether it is possible to inhibit glucuronidation on a transient basis before administration of glucuronidatable drugs so as to increase markedly the uptake of free (active) drug. To assess whether the mouse is a valid model for targeting downregulation of UGTs through the disruption of phosphorylation, we showed that mouse recombinant UGT1A7 metabolizes mycophenolic acid (MPA) and that curcumin treatment of the isozyme reversibly inhibits MPA glucuronidation in a concentration- and a time-dependent manner. Based on the UGT isozymes' requirement for phosphorylation, we demonstrated transient downregulation of glucuronidation of UGTs by pretreating antigen-stimulated mice with dietary curcumin and then administering highly glucuronidatable MPA. We observed a six- to eight-fold increase in both free circulating MPA and immunosuppression. MPA is widely used to treat renal transplant patients because of its selective inhibition of the enzyme supporting activated T- and B-lymphocyte proliferation. To verify the effects of curcumin on UGT phosphorylation and drug efficacy, we found the following: (1) concentration-dependent inhibition of recombinant UGTs and of those distributed in mouse gastrointestinal (GI) mucosa by the PKC kinase inhibitors curcumin or calphostin-C elicits transient or permanent UGT inhibition, respectively; (2) curcumin downregulates [³³P]orthophosphate incorporation into immunoprecipitable UGTs in colon LS180 cells; (3) curcumin is responsible for 95 percent inhibition, thus allowing sufficient curcumin glucuronidation to initiate reversal of inhibition; and (4) curcumin injection into mouse duodenal loops or its oral pre-administration to mice before MPA administration increases free MPA in blood and GI tissues six- to eight-fold, with similar effects on MPA immunosuppression of antigen-treated mice. The evidence demonstrates that UGT phosphorylation is a target for transient downregulation of glucuronidation, allowing concurrent therapeutic drug administration to elicit increased drug bioavailability.

Basu NK, Kole L, Kubota S, Owens IS. Human UDP-glucuronosyltransferases show atypical metabolism of mycophenolic acid and inhibition by curcumin. Drug Metab Dispos 2004;32:768-73.

¹ Amanda Garza, BS, former Predoctoral Fellow
² Chimere Mbas-Jones, former Summer Student
³ Elaing Change, former Summer Student
⁴ Matthew Pennington, former Summer Student
⁵ Labanyamoy Kole, PhD, former Postdoctoral Fellow

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.