Alteration of the Function of the UDP-Glucuronosyltransferase 1A Subfamily by Cytochrome P450 3A4: Different Susceptibility for UGT Isoforms and UGT1A1/7 Variants

Abstract

Functional protein-protein interactions between UDPglucuronosyltransferase (UGT)1A isoforms and cytochrome P450 (CYP)3A4 were studied. To this end, UGT1A-catalyzed glucuronidation was assayed in Sf-9 cells that simultaneously expressed UGT and CYP3A4. In the kinetics of UGT1A6-catalyzed glucuronidation of serotonin, both Michaelis constant (K m ) and maximal velocity (V max ) were increased by CYP3A4. When CYP3A4 was coexpressed with either UGT1A1 or 1A7, the V max for the glucuronidation of the irinotecan metabolite (SN-38) was significantly increased. S 50 and K m both which are the substrate concentration giving 0.5 V max were little affected by simultaneous expression of CYP3A4. This study also examined the catalytic properties of the allelic variants of UGT1A1 and 1A7 and their effects on the interaction with CYP3A4. Although the UGT1A1-catalyzing activity of 4-methylumbelliferone glucuronidation was reduced in its variant, UGT1A1*6, the coexpression of CYP3A4 restored the impaired function to a level comparable with the wild type. Similarly, simultaneous expression of CYP3A4 increased the V max of UGT1A7*1 (wild type) and *2 (N129K and R131K), whereas the same was not observed in UGT1A7*3 (N129K, R131K, and W208R). In the kinetics involving different concentrations of UDPglucuronic acid (UDP-GlcUA), the K m for UDP-GlcUA was significantly higher for UGT1A7*2 and *3 than *1. The K m of UGT1A7*1 and *3 was increased by CYP3A4, whereas *2 did not exhibit any such change. These results suggest that (1) CYP3A4 changes the catalytic function of the UGT1A subfamily in a UGT isoformspecific manner and (2) nonsynonymous mutations in UGT1A7*3 reduce not only the ability of UGT to use UDP-GlcUA but also CYP3A4-mediated enhancement of catalytic activity, whereas CYP3A4 is able to restore the UGT1A1*6 function.

Suppression of Cytochrome P450 3A4 Function by UDP-Glucuronosyltransferase 2B7 through a Protein-Protein Interaction: Cooperative Roles of the Cytosolic Carboxyl-Terminal Domain and the Luminal Anchoring Region

“…Although many pairs of P450-UGT complexes can be formed (Ishii et al, 2007), we have particularly focused on a CYP3A4-UGT2B7 interaction, because these enzymes are involved in the metabolism of many drugs. The association of CYP3A4 and UGT2B7 has been confirmed by several biochemical techniques including coimmunoprecipitation and cross-linking (Takeda et al, , 2009Ishii et al, 2010Ishii et al, , 2014. Because CYP3A4 alters the regio-selectivity of UGT2B7-catalyzed morphine glucuronidation ) the binding of CYP3A4 and UGT2B7 is undoubtedly a functional interaction.…”

“…After PstI treatment, CNX cDNA was also cloned into pFastBac1. The construction of pFastBac1-CYP3A4 was carried out as previously described (Ishii et al, 2014). For the pull-down assay, CYP3A4 having a carboxyl terminus tagged with hexahistidine (His-CYP3A4) was constructed by PCR.…”

“…In the case of expression of CYP3A4, 1 mg/ml hemin/1.25% albumin complex was added to the medium at a concentration of 1.4 mM hemin 24 hours after transfection. Microsomes were prepared from the transfected cells according to the protocols described previously (Ishii et al, 2014).…”

“…However, there are likely to be a number of P450-UGT interactions involving a variety of P450 and UGT isoforms (Thummel and Wilkinson, 1998;Ishii et al, 2010Ishii et al, , 2014, and their effects on P450s have not yet been elucidated. Further studies are necessary to understand the details and physiologic significance of these P450-UGT interactions.…”

Altered CYP2C9 Activity Following Modulation of CYP3A4 Levels in Human Hepatocytes: an Example of Protein-Protein Interactions

“…This modulation could be physiologically advantageous based on the roles of these enzymes in the sequential metabolism of xenobiotics. Recently, Ishii et al (2014) examined possible effects of CYP3A4 on UGT1A isoforms and concluded that the presence of CYP3A4 led to increases in UGT1A1 and UGT1A7 activities, while observing differential effects on the activities of allelic variants of these UGT isoforms (Ishii et al, 2014).…”