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 liver, kidney, the 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, and dihydrotestosterone, and dietary aromatic-like therapeutics to 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/PKCε–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) residues
Prostate-distributed mouse Ugt2b34 and Ugt2b36 control estrogenic metabolites.
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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 stratification of human prostate basal/luminal cells with intervening gap junctional structures that likely allow movement of small molecules between the two cells.
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 Km UGT-2B7 (14M), Ugt-2b34 and Ugt-2b36 metabolized 4-hydroxyestrone with 90M Km and 430M Km, respectively. 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 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 (Raychoudhuri A et al., Biosci Rep 2015;in press).
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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 Analyses for Caspases 8/3 activities. Cellular material from COS-1 (A, B) and PC3 (C) transfected cells were incubated for glucuronidaton for DHT and/or its metabolite (Andro) for 1-3h at 37º C (2A, B, C) and Western Blot analysis were carried out using antu-Caspase8, anti-Caspase3, anti-Src, anti-UGT-1168 or anti-β Actin antibodies.
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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) + CK5 + CK14 + 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.
