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MOLECULARBIOLOGY,
REGULATION, AND BIOCHEMISTRY OF
UDP-GLUCURONOSYLTRANSFERASEISOZYMES
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Ida S. Owens, PhD, Head, Section on Genetic Disorders of
Drug Metabolism Nikhil K. Basu, PhD, Research Fellow Bhabadeb Chowdhury, PhD, Research Fellow Rajat Banerjee, PhD, Visiting Fellow Partha Mitra, PhD, Visiting Fellow Amanda Garza, BS, Predoctoral
Fellow Matthew Pennington, Summer Student |
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Mammalian UDP-glucuronosyltransferase (UGT) isozymes
facilitate detoxification of endogenous metabolites and numerous potentially
injurious lipid-soluble phenols derived from the diet and the environment.
Isozymes detoxify by attaching glucuronic acid to metabolites, drugs, toxins,
and environmental chemicals, generating water-soluble excretable products.
Glucuronidation reactions prevent accumulation of neurotoxic levels of plasma
bilirubin, inactivate many drugs, and avert mutagenicity and carcinogenicity
of aromatic hydrocarbons, including benzo(a)pyrene, which is found in
cigarette smoke and automobile emissions. Moreover, UGTs prevent accumulation
of dietary phenols that inhibit enzymes. Conversely, extensive
glucuronidation can be disadvantageous. The premature clearance of many
orally administered therapeutic drugs is a long-standing problem and is
associated with UGT metabolism. Such metabolism is overcome by administering
compensatory higher doses that can lead to serious side effects. Thus, for
decades, drug inefficiency has been the impetus for developing UGT
inhibitor(s). The enzymatic mechanism(s) and properties that enable a limited
number of UGTs to convert numerous structurally diverse lipid-soluble phenols
to innocuous glucuronides are unknown. Therefore, an important research aim
is to understand the properties and mechanism(s) that enable detoxification
of a vast number of agents in order to maintain chemical homeostasis. Phosphorylation of UGT proteins with signaling Basu,
Mitra, Kole,a Kubotab While analyzing UGTs to understand glucuronidation
requirements in human colon cell lines, we discovered that the isozymes
require phosphorylation that is regulated via signaling. While
phosphorylation is mediated by certain PKC isozymes or tyrosine kinase(s),
the action of classical PKC agonists and antagonists as well as the effects
of PKC translocation–specific inhibitor peptides confirmed the
involvement of signaling events. Furthermore, immunocomplexing of UGT from
solubilized human LS180 colon cells with antiUGT traps specific PKC isozymes
and associated receptors that are used for translocation to membranes.
Parallel hydrogen-peroxide (H2O2)-stimulation of UGT
phosphorylation and glucuronidation activity and inhibition of H2O2-enhanced
activity by catalase and herbimycin demonstrated that cellular oxidants are
signal(s) for the PKC-UGT system. Specifically, our results demonstrated that
UGT1A1, 1A7, and 1A10 undergo phosphorylation by PKC(s), which are, in turn,
phosphorylated and activated by tyrosine kinase(s). Given that the general kinase
inhibitor curcumin and the highly specific PKC inhibitor calphostin-C
inhibited each of 10 different UGTs independently transfected into COS-1
cells, our results suggest that phosphorylation is a universal requirement
for glucuronidating enzymes. For the UGT2B family
members that generally metabolize endogenous substrates, we uncovered
evidence for direct phosphorylation by tyrosine kinase. In addition to three
PKC phosphorylation sites, UGT2B7 has two tyrosine phosphorylation sites.
Alteration of one or all PKC sites in UGT2B7 that generated a triple mutant
had no effect on activity while a single or double mutation at tyrosine sites
abrogated activity. Both herbimycin-C and genistein are tyrosine kinase
inhibitors; UGT1A1, 1A7, and 1A10 were sensitive to the former and UGT2B7
more sensitive to the latter agent. In summary, our results demonstrate that all
tested UGTs undergo phosphorylation and that UGT1A family members are
phosphorylated via PKCs, with signaling mediated by oxidative stress. At least
one UGT2B family member requires tyrosine phosphorylation. Overexpression of
PKCepsilon in conjunction with UGT1A7 or its mutants indicated that
PKCepsilon phosphorylates only at position 432, whereas in vitro
studies with 1A7-specific peptides compared with mutant peptides demonstrated
that at least PKCalpha, betaII, or epsilon can phosphorylate at positions 73,
202, and 432 in UGT1A7 and 1A10. Mutants at all positions in UGT1A1, 1A7, and
1A10 were null, except mutants at the common position 435/432, indicating
that phospho-group(s) play a unique and novel role in catalysis by
controlling pH optima and substrate selection. S432G-1A7 revealed a dramatic
shift in its pH optimum from 8.5 to 6.4 in parallel with the acquisition of
new substrate activities or loss of activities resembling 1A10 substrate
selections. 17beta-estradiol was among the new in vitro and in
cellulo substrates for S432G-UGT1A7–transfected cultures, but not
for 1A7-transfected cells. The new substrate profile resembled that for
wild-type 1A10 and its S432G mutant. Although we confirmed that PKCepsilon
overexpression positively affects only phosphoserine-432 in 1A7 and that
S432G-1A7 mutant acquires new activity, a remaining question is whether
inhibitors of PKC(s) that phosphorylate serine-432 also support
17beta-estradiol glucuronidation. Treatment of 1A7-transfected COS-1 cells
with PKCeta-specific translocation-inhibitor peptide revealed an 11-fold
concentration-dependent increase in 17beta-estradiol-glucuronide production
in parallel with decreasing levels of phosphoserine-432. Together with our
demonstration of progressive inhibition of eugenol activity (pH 8.0), we
showed an increase in 17beta-estradiol-glucuronide (pH 7.0); both events are
associated with the pH shift. Moreover, treatment of 1A7-transfected cells
with increasing concentrations of general the PKC inhibitors
bisindolylmaleimide (BIM) or chelerythrine caused 11- and nine-fold increases
in 17beta-estradiol-glucuronide production, respectively, in parallel with
decreases in phosphoserine-432. Even modest decreases in phosphoserine-432
following treatment with the classical PKC inhibitor Gö-6976 caused a
remarkable three- to five-fold increase in 17beta-estradiol-glucuronide
production. The prominent increase in 17beta-estradiol glucuronidation in
parallel with dephosphorylation of serine-432 demonstrates that inhibition of
select PKC isozymes can have a significant effect on UGT substrate
specificity and that dephosphorylation of serine-432 is responsible for the
expanded substrate activity. Basu NK, Kole L, Owens IS. Evidence for
phosphorylation requirement for human bilirubin UDP-glucuronosyltransferase
(UGT1A1) activity. Biochem Biophys Res Commun 2003;303:98-104. Basu NK, Kubota S,
Meselhy MR, Ciotti M, Chowdhury B, Hartori M, Owens IS. Gastrointestinally
distributed UDP-glucuronosyltransferase 1A10, which metabolizes estrogens and
nonsteroidal anti-inflammatory drugs, depends upon phosphorylation. J Biol
Chem 2004;279:28320-28329. Enhanced control of gastrointestinal
chemical absorption by location and properties of UGT Basu,
Mitra, Kolea; in collaboration with McDonagh Using Northern blot, in situ, and
biochemical analyses, we determined the distribution and function of the
bilirubin metabolizing isozymes UGT1A1, 1A7, 1A8, 1A9, and 1A10 and, for the
first time, found them to be segmentally distributed throughout the GI tract
in mucosal epithelia. Recombinant isozymes
exhibited a pH optimum of 6.4, pH 7.6 or a broad range; increasing substrate
concentrations either did not affect or progressively inhibited activity. Under
different optimal conditions, all enzymes exhibited wide substrate selections
for dietary and environmental chemicals. The impact of glucuronidation on drug
and chemical absorption at the GI level was demonstrated by exploiting our
recent finding that UGTs require phosphorylation, with [33P]orthophosphate
incorporation into immunoprecipitable UGTs confirming phosphorylation. The
simultaneous loss of UGT radiolabeling and activity in cell lines by the
general kinase inhibitor curcumin and the PKC-specific inhibitor calphostin-C
likewise confirmed the requirement for phosphorylation. Using
curcumin-treated mouse duodenal loops, we demonstrated that free bilirubin (a
marker) uptake dramatically increased concurrently with decreases in
bilirubin-glucuronides in portal blood, lumen, and tissue. Our results
represent the first direct evidence that UGTs control GI absorption of
polyphenols and that maintenance of their phosphorylation is required to
limit chemical absorption. Basu NK, Ciotti M, Hwang MS, Kole L, Mitra PS,
Cho JW, Owens IS. Differential and special properties of the major human UGT1-encoded
gastrointestinal UDP-glucuronosyltransferases enhance potential to control
chemical uptake. J Biol Chem 2004;279:1429-1441. Structural analysis of UGT Banerjee,
Basu, Pennington Because UDP-glucuronosyltransferases are bound
to the membrane of the endoplasmic reticulum and are thus difficult to purify
for crystallization, structural analysis of these critical detoxifying
isozymes has posed a challenge. Importantly, defective UGT isozymes are
responsible for the lethal childhood Crigler-Najjar disease and therapeutic
drug toxicities. Whereas all previous attempts to find structurally
determined proteins homologous to the UGT1 isozymes have failed, Matthew
Pennington carried out computer- and homology-based molecular modeling
searches of the Protein Data Base (PDB) for structural matches of UT1A10. He
used a Silicon Graphics O2 workstation and Insight II software
(Molecular Simulation, Inc.) with the Homology, MODELER, Discover,
Bioploymer, and SeqFold expansion modules. The first modules are necessary
for all homology or protein work. The SeqFold module is a sequence homology
search engine that uses a threading technique to identify potentially
homologous proteins based on the predicted secondary structure of the target
sequence and known secondary structures within structurally solved protein
databases. Pennington performed all searches against a nonredundant version
of the Brookhaven PDB and created a protein homolog by using, first, SeqFold
and, second, the structure of that sequence homolog to map the sequence of an
unknown structure to a set of three-dimensional coordinates. Initially, he
performed the application on protein regions near the core and on
well-conserved sections. He then built regions of poor conservation onto the
core by using a fragment database and conformation searching techniques. He
next optimized the initial model by using a simulated-annealing technique.
Because 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 and selected the new secondary-structure
prediction-based search engine SeqFold, which is proficient in identifying
structural homologs low on direct sequence-sequence identity to the target
protein. Although SeqFold uncovered many low-identity homologs that bound
similar substrates to UGT, the most prominent one with the most chemically
proper structure was a UDP-galactosegUDP-glucose isomerase, known in the PBD
as 1XEL. He located highly homologous regions of UGT1A10 and 1XEL and
confirmed them to be those involved in binding UDP-glucose, clearly analogous
to UDP-glucuronic acid (UDPGA). Pennington identified primarily two lysines
(positions 314 and 315) and one asparagine (position 292) predicted to be
critical in identifying the uracil-diphosphate portion of the donor
substrate, UDPGA. Our recent studies showed that mutants at 314 or 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, either
was not affected or had a sharper pH optimum. We will now mutate the
identified conserved asparagine. The results indicate that lysines 314 and
315 are critical residues. Improvement of mycophenolic acid
immunosuppressant activity by transient inhibition of gastrointestinal UGT
activity Basu,
Kole,a Chowdhury While the promising immunosuppressant
mycophenolic acid (MPA) is being widely prescribed for both adult and child
renal transplant patients and for autoimmune diseases, serious side effects
are associated with high dosage requirements as a result of extensive
glucuronidation. We found that the cellular distribution and biochemical
properties of UGT1A7, 1A8, 1A9, and 1A10, the primary metabolizers of MPA,
contribute significantly to high oral-dose requirements. In situ
hybridization studies revealed that UGT1A7-, 1A9-, and 1A10-mRNAs are located
in GI mucosal epithelia; studies with microsomes isolated from adjacent
specimens showed that esophagus, ileum, duodenum, and colon have moderately
high to high glucuronidating activities. 1A7 and 1A8 metabolized MPA
maximally at pH 7.5 while activity for 1A9 and 1A10 was maximum at pH 6.4.
Recombinant UGTs avidly glucuronidated MPA, showing nearly linear increases
in activity until concentrations reached between 0.800 and 1.5 mM; UGT1A9
reached maximum activity at 2.5 mM. At maximum activity, 1A7 through 1A9
generated between 20 and 27 percent acylglucuronide while 1A10 generated 37
percent. To establish the in vivo impact of MPA glucuronidation, we
used the general kinase inhibitor curcumin and the highly specific protein
kinase C inhibitor calphostin-C, which target the newly discovered
phosphorylation requirement, on LS180 colon cells, to inhibit UGTs. Transient
inhibition of glucuronidation by oral pretreatment with curcumin before MPA
administration caused a six-fold increase in immunosuppression of
antigen-stimulated spleen cytotoxic T-lymphocyte proliferation in mice.
Hence, glucuronidation can adversely affect drug efficacy. Moreover, curcumin
pretreatment to inhibit GI-distributed UGTs represents a model for increasing
bioavailability of highly glucuronidated drugs. Basu NK, Kole L, Kubota S, Owens IS. Human
UDP-glucuronosyltransferases show atypical stimulation by mycophenolic acid
and inhibition by curcumin. Drug Metab Disp 2004;32:768-773. aLabanyamoy
Kole, PhD, former Postdoctoral Fellow bShigeki COLLABORATORS Masahiko Negishi, PhD, Laboratory
of Reproductive and Developmental Toxicology, NIEHS, For
further information, contact owens@helix.nih.gov |
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