Molecular Biology, Regulation, and Biochemistry of UDP–Glucuronosyltransferase Isozymes
- Ida S. Owens, PhD, Head, Section on Genetic Disorders of Drug Metabolism
UDP–glucuronosyltransferase (UGT) isozymes–distributed primarily in the liver, kidney, gastrointestinal tract, and steroid-responsive tissues–are known to carry out the essential function of converting innumerable structurally diverse lipophilic endogenous substrates, such as neurotoxic bilirubin, catechol estrogens, dihydrotestosterone, and dietary aromatic-like therapeutics into water-soluble excretable glucuronides. Most importantly, environmental pro-carcinogens and contaminants derived from pyrolysates are converted to avoid chemical toxicities. Our studies demonstrated that each UGT isozyme so far examined requires on-going regulated phosphate signaling, which enables an active site to convert an unspecified number of substrates. Recently, further studies showed that the human prostate luminal-cell UGT-2B15 and basal-cell UGT-2B17, which are 97% identical, have an additional Src or Src/PKCepsilon–partnership phosphorylation site, respectively, at position 98–100. We found that the two isozymes exhibit opposite behavior when their Src sites are compromised: UGT-2B15 becomes polyubiquitinated, thus exhibiting a pro-apoptotic effect, while the activity of UGT-2B17 is elevated by 50%. Our studies will thus continue to detail and understand the specific reactions involved in human prostate luminal-cell apoptosis and de-ubiquitination. In collaboration with ongoing research within the NCI, NIDCR, and with researchers at the University of Maryland, we will also carry out basic studies to better understand prostate cancer development.
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Figure 1. Mass-spectrometric analysis of TpYpSp phosphorylation of UGT-2B17
Phosphorylation of UGT-2B17 at 98–100 (TYS—threonine/tyrosine/serine) residues
Top: TY Phosphorylation. Mass spectrometry analysis of phosphorylation site showing TY at positions 98-99 in UGT-2B17. In the table: red color means match to b ions, blue color matches with y ions.
Bottom: YS Phosphorylation. Mass spectrometry analysis of phosphorylation site showing YS at positions 99-100 in UGT-2B17.
Pro- and anti-apoptotic sequences control human prostate DHT–metabolizing UGT-2B15 and UGT-2B17
Similar to each of the six of 19 UGTs examined, UGT-2B17 requires PKCalpha–mediated phosphorylation signaling at position 172, which enables catalysis of an unspecified number of substrates. Moreover, mass spectrometry confirmed that UGT-2B17 has the triple-phosphorylated sequence threonine/tyrosine/serine (TYS) at positions 98–100. Notably, analyses of anti-PKCalpha–immunocomplexed wild-type (wt) UGT-2B17 and its 4-phosphorylated anti-PKCepsilon–immunocomplexes of UGT-2B17 show a dense smearing pattern, except in the case of its Y99F (tyrosine→phenylalanine) mutant or following expression of constructs in Src-kinase–free cells. The smearing is also consistent with robust signaling surrounding the Src/PKCepsilon–partnership phosphorylation site. Following UGT-2B17 expression in Src–/– compared with Src+/– cells, glucuronidation of dihydrotestosterone (DHT) and its 3α-androstane-5α,17β-diol (ADT-diol) metabolite indicates that Src inhibits the glucuronidation by around 50%, which necessarily concomitantly elevates anti-apoptotic DHT levels. Following the exchange of isoleucine/tyrosine/glycine (IYG) in wild-type (wt) UGT-2B15(IYG) and TYS in wt UGT-2B17(TYS) at their comparable positions of 98 through 100 and subsequent transfection into COS-1 cells, UGT-2B17(IYG) generated 10-fold greater activation in cellulo of caspases 8/3 than did wt UGT-2B15, while mutant UGT-2B15(TYS) suppressed activation of caspases 8/3 more than 50% compared with UGT-2B15 levels. The evidence thus indicates that the triple-phosphorylated TpYpSp site on UGT-2B17 creates a signaling site involving Src and PKCepsilon that is anti-apoptotic, while the Src–specific binding/phosphorylation site at position 98–100 in UGT-2B15 is pro-apoptotic. The evidence also indicates that serine 172 is phosphorylated to carry out signaling-mediated catalysis, as shown for some seven out of 19 other human UGTs.
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Figure 2. Predicted small-molecule movements and enzymatic reactions in prostate epithelial cells
Distribution of normal prostate steroidogenic and UGT isozymes as presented in our publication, S. K. Chakraborty et al., J Biol Chem 2012;287:24387. Based on studies cited, prostate DHT synthesis and its metabolism are summarized in the schematic. The EM shows 1:1 strati cation of human prostate basal/luminal cells with intervening gap junctional structures that likely allow movement of small molecules between the two cells.
Image taken from Reference 5.
Whereas prostate biochemists discovered that, by occupying the androgen receptor, DHT plays a vital role in supporting the synthesis of the more than 200 secretory proteins necessary for sperm transport fluid, we established that the critically important UGT-2B15 is required for the luminal-cell conversion of DHT to a water-soluble DHT–glucuronide and for the overall maintenance of the luminal cell. More recently, prostate biochemists established that basal cell–distributed UGT-2B17 metabolizes DHT at 10 to 20-fold higher rates than does UGT-2B15. While the exact role of UGT-2B17 in the prostate basal cell remains unknown, studies indicate that the isozyme functions primarily in a supporting role to ‘house’ intermediate stem cells that contain both cytokeratin cell-surface markers for both luminal and basal cells in a ‘Basal Cell Compartment.’ Thus, the robust activity of UGT-2B17 is thought to play a role in protecting both newly generated luminal and basal cells for prostate continuity.
While the human prostate luminal cell–distributed UGT-2B15 supports regulated phosphorylation for two different functions, at least one phosphate per UGT-2B15 isozyme undergoes ongoing phosphate signaling at a non-fixed active site, which enables catalysis of an unspecified number of substrates, according to 6 of the 19 human UGTs examined.
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Figure 3. Controlling sequences for apoptosis
Controlling sequences for pro-apoptosis and anti-apoptosis in UGT-2B15 and UGT-2B17 expressed in COS-1 cells and the aggressive PC3 prostate cell line
Glucuronidation of the Androgens and Immunoblot Analysis for Caspases 8/3 activities. Cellular material from COS-1 (A,B) and PC3 (C) transfected cells were incubated for glucuronidation for DHT and/or its metabolite (Andro) for 1-3h at 37°C (2A,B,C) and Western Blot analysis were carried out using anti-Caspase8, anti-Caspase3, anti-Src, anti-UGT-1168 or anti-β Actin antibodies
Prostate-distributed mouse Ugt2b34 and Ugt2b36 control estrogenic metabolites.
To establish an in vivo mammary-gland model that prevents depurination by 4-OH-catecholestrogens associated with the initiation of carcinogenesis, we pursued studies to identify mouse homologs of the highly effective human UGT-2B7. Using sequence analysis, we found that mouse Ugt-2b34 and Ugt-2b36 homologs avidly metabolize the test agent 4-hydroxyestrone, with Ugt-2b35 expressing trivial activity. Unlike low-K m UGT-2B7 (14M), Ugt-2b34 and Ugt-2b36 respectively metabolized 4-hydroxyestrone with 90M K m and 430M Km. Unexpectedly, the mouse isozymes are distributed primarily in male hormone–responsive tissues, whereas human UGT-2B7 is found primarily in female hormone–responsive tissues. Also, we found that Ugt-2b34 metabolizes the non-classical estrogenic DHT metabolite ADT-diol at a greater rate than DHT, which is not known to be estrogenic. Notably, UGT-2B7 does not metabolize xeno-estrogens; Ugt-2b34 and Ugt-2b36 did, however, metabolize bisphenol A (BPA) and diethylstilbestrol (DES) at superior rates. We also found, through real-time PCR–based analysis of estrogen receptor alpha (Esr1) gene knockout in mouse prostate, 50% and 63% lower Ugt2b34 mRNA and Ugt2b36 mRNA levels, respectively, than in controls. However, estrogen receptor beta (Esr2) knockout (KO) revealed a 2.7/3.3-fold increase in Ugt-2b34 mRNA and Ugt-2b36 mRNA, respectively, in the prostate. Esr1 KO completely suppressed Ugt-2b34 and Ugt-2b36 mammary-gland mRNA; Esr2 KO caused a 12-fold increase in Ugt-2b34 mRNA without affecting Ugt-2b36 mRNA. Hence, according to tissue-distribution studies, it appears that male mice benefit from both Ugt isoforms, while females benefit from only one Ugt. Our findings for Ugt-2b34 and Ugt-2b36 suggest that the two mouse isozymes are intrinsically programmed to protect against a more complex environment than are human high-activity UGT-2B7 and low-activity UGT-2B4 isozymes.
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Figure 4. Diagrammatic representation of normal prostate epithelial and associated cells
Normal prostate epithelial and associated cells with UGT-2B15, UGT-2B17, and cell-surface markers.
Diagrammatic representation of normal prostate epithelial and associated cells. Representations of normal human prostate luminal and basal epithelial cells surrounded by primary and intermediate-stem, neuroendocrine, normal fibroblast cells and separated by the basement membrane. A fraction of the progenitor intermediate stem cell population contains cytokeratin cell-surface markers for both (luminal) and basal [(CK8+Ck18) + CK 5 + CK 14 + P63] cells, respectively. Authors of these studies suggest this is evidence the two distinct prostate epithelial cells derive from a single stem cell that ultimately gives rise to two distinct differentiated epithelial cells.
Publications
- 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.
- 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.
- Mitra PS, Basu NK, Basu M, Chakraborty S, Saha T, Owens IS. Regulated phosphorylation of a major UDP-glucuronosyltransferase isozyme by tyrosine kinases dictates endogenous substrate selection for detoxification. J Biol Chem 2011 286:1639-1648.
- Chakraborty SK, Basu NK, Jana S, Basu M, Raychoudhuri A, Owens IS. Protein kinase Calpha and Src kinase support human prostate-distributed dihydrotestosterone-metabolizing UDP-glucuronosyltransferase 2B15 activity. J Biol Chem 2012 287:24387-24396.
Collaborators
- Praveen Arany, BDS, MDS, MMSc, PhD, Oral and Pharyngeal Cancer Branch, NIDCR, Bethesda, MD
- James L. Gulley, MD, PhD, FACP, Genitourinary Malignancies Branch, Center for Cancer Research, NCI, Bethesda, MD
- Antony McDonagh, PhD, University of California San Francisco, San Francisco, CA
- Zhihong Nie, PhD, Maryland Nanocenter, University of Maryland, College Park, MD
- Juan Rivera, PhD, Molecular Immunology and Inflammation Branch, NIAMS, Bethesda, MD
Contact
For more information, email owens@helix.nih.gov.
