Evidence for phosphorylation requirement for human bilirubin UDP-glucuronosyltransferase (UGT1A1) activity

Kole

Biochemical and Biophysical Research Communications

et al. 2007

Self Cite

Finding UDP-glucuronosyltransferases (UGT) require protein kinase C-mediated phosphorylation is important information that allows manipulation of this critical system. UGTs glucuronidate numerous aromatic-like chemicals derived from metabolites, diet, environment and, inadvertently, therapeutics to reduce toxicities. As UGTs are inactivated by downregulating PKCs with reversiblyacting dietary curcumin, we determined the impact of gastro-intestinal glucuronidation on free-drug uptake and efficacy using immunosuppressant, mycophenolic acid (MPA), in mice. Expressed in COS-1 cells, mouse GI-distributed Ugt1a1 glucuronidates curcumin and MPA and undergoes irreversibly and reversibly dephosphorylation by PKC-specific inhibitor calphostin-C and generalkinase inhibitor curcumin, respectively, with parallel effects on activity. Moreover, oral curcuminadministration to mice reversibly inhibited glucuronidation in GI-tissues. Finally, successive oraladministration of curcumin and MPA to antigen-treated mice increased serum free-MPA and immunosuppression up to 9-fold. Results indicate targeted inhibition of GI-glucuronidation in mice markedly improved free-chemical uptake and efficacy using MPA as a model.Whereas the primary role of UDP-glucurononsyltransferases (UGT) is to detoxify numerous structurally diverse lipophilic chemicals, including metabolites, dietary constituents, environmental toxicants, carcinogens and, inadvertently, therapeutic chemicals, there is a limited capacity to manipulate glucuronidation to improve protection or reduce premature ‡ Corresponding authors at: National Institutes of Health, Building 10, Room 8D-42, Bethesda, MD 20892-1830, E-mail addresses, telephone and fax numbers: owensi@mail.nih.gov; 301-496-6091, and 301-480-8042; basun@mail.nih.gov, 301-496-8825, 301-451-4288. Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. Whereas MPA readily forms ether-linked-(MPAG) or acyl-glucuronides (AcMPAG) [9-11], we studied MPA glucuronidation in vitro with human UGTs and found 4 isozymes are avid metabolizers [12] that reach saturation kinetics between 1.6 and 2.4 mM with Km values between 0.25 and 0.55 mM. These UGT isozymes, encoded at the UGT1 complex locus, were found strategically and differentially expressed in the gastrointestinal (GI) mucosa [1]. In this report, we established under both in-vitro and in-vivo conditions that mouse Ugt1a1 also requires phosphorylation, which can be transiently downregulated by nontoxic kinase inhibitor, curcumin [2,3,13], and irreversibly by calphostin-C, similar to that for human UGTs [2,3]. Moreover, effects of...

“…In addition Ugt1a1 has 3 computer-detected serine-specific PKC phosphorylation sites at position 203, 437, and 529, similar to human isozymes [3].…”

Section: Sensitivity Of Mouse Ugt1a1 To Curcumin and Calphostin-cmentioning

confidence: 99%

Section: Sensitivity Of Mouse Ugt1a1 To Curcumin and Calphostin-cmentioning

confidence: 99%

mentioning

confidence: 97%

mentioning

confidence: 99%

mentioning

confidence: 99%

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Targeted inhibition of glucuronidation markedly improves drug efficacy in mice—A model

Kole

Biochemical and Biophysical Research Communications

et al. 2007

Self Cite

Regulation of uridine diphosphate‐glucuronosyltransferase 1A3 activity by protein phosphorylation

Xiao

Yao

Jiang

et al. 2015

Biopharm & Drug Disp

Protein phosphorylation is a vital post-translational modification. This study investigated the effect of phosphorylation on human uridine diphosphate (UDP)-glucuronosyltransferase 1A3 (UGT1A3) activity. Curcumin and calphostin C suppressed the activity and phosphorylation of recombinant UGT1A3 expressed in Sf9 cells. These results indicate that UGT1A3 undergoes phosphorylation, which is required for its catalytic activity. Calphostin C is a highly specific protein kinase C (PKC) inhibitor, so three predicted PKC phosphorylation sites in UGT1A3 were examined. Site-directed mutation analysis at residues 28, 43 and 436 (from serine to glycine) was conducted. Compared with the wild-type, the S43G-mutant showed significantly decreased UGT1A3 catalytic activity. Furthermore, the UGT1A3 activity of wild-type and S43G-mutant was down-regulated by calphostin C, whereas the calphostin C inhibitory effect was much weaker on the S43G-mutant than the wild-type. In conclusion, phosphorylation plays an important role in UGT1A3 activity, and the serine at site 43 in UGT1A3 is most likely a phosphorylation site.

Reply:

Poorten

George

2008

Hepatology

Reply:We thank Drs. Dogru and Teoman for their interesting comments regarding our recent article on the importance of visceral fat to the pathogenesis of nonalcoholic steatohepatitis. 1 Our study showed visceral fat to be a key mediator of steatohepatitis independent of insulin resistance and importantly, independent of a diagnosis of metabolic syndrome (MetS). We agree that all components of the MetS can variably be associated with both insulin levels and those of circulating adipokines. However, the MetS (as defined by various criteria) has been developed purely for operational reasons. This syndrome is the consequence, rather than the cause of metabolic dysregulation. For this reason, in our multivariate analysis, we controlled the effects of visceral fat for both circulating adipokines and insulin sensitivity as measured by the homeostasis model assessment of insulin resistance (HOMA-IR). The HOMA-IR has been shown to correlate well with the results of the euglycemic-hyperinsulinemic clamp in patients with and without type 2 diabetes, even in the presence of metformin or sulfonylureas. 2,3 Thus, any confounding effect of MetS components, or indeed the medications used to treat these components, will have been reflected and controlled for by HOMA-IR levels and those of the circulating adipokines.We are happy to provide some additional results that clarify the relationship between visceral fat and the other components of the MetS (Table 1). These demonstrate that the most marked changes in visceral fat occur with increasing dysregulation of glucose handling, while those associated with dyslipidemia and hypertension did not reach statistical significance. When the association between these individual metabolic variables and necroinflammation and fibrosis in nonalcoholic steatohepatitis was considered, only MetS glucose (P ϭ 0.04) was significant, while elevated blood pressure (P ϭ 0.35), elevated triglycerides (P ϭ 0.9), and low high-density lipoprotein (P ϭ 0. 13) were not associated. Given that we have already specifically controlled for the effects of insulin sensitivity, we did not further analyze the data.DAVID VAN