Abstract

   The conjugative metabolism mediated by UDP-glucuronosyltransferase
   enzymes (UGTs) significantly influences the bioavailability and
   biological responses of endogenous molecule substrates and xenobiotics
   including drugs. UGTs participate in the regulation of cellular
   homeostasis by limiting stress induced by toxic molecules, and by
   controlling hormonal signaling networks. Glucuronidation is highly
   regulated at genomic, transcriptional, post-transcriptional and
   post-translational levels. However, the UGT protein interaction
   network, which is likely to influence glucuronidation, has received
   little attention. We investigated the endogenous protein interactome of
   human UGT1A enzymes in main drug metabolizing non-malignant tissues
   where UGT expression is most prevalent, using an unbiased proteomics
   approach. Mass spectrometry analysis of affinity-purified UGT1A enzymes
   and associated protein complexes in liver, kidney and intestine tissues
   revealed an intricate interactome linking UGT1A enzymes to multiple
   metabolic pathways. Several proteins of pharmacological importance such
   as transferases (including UGT2 enzymes), transporters and
   dehydrogenases were identified, upholding a potential coordinated
   cellular response to small lipophilic molecules and drugs. Furthermore,
   a significant cluster of functionally related enzymes involved in fatty
   acid β-oxidation, as well as in the glycolysis and glycogenolysis
   pathways were enriched in UGT1A enzymes complexes. Several partnerships
   were confirmed by co-immunoprecipitations and co-localization by
   confocal microscopy. An enhanced accumulation of lipid droplets in a
   kidney cell model overexpressing the UGT1A9 enzyme supported the
   presence of a functional interplay. Our work provides unprecedented
   evidence for a functional interaction between glucuronidation and
   bioenergetic metabolism.

   Keywords: UGT, proteomics, protein-protein interaction, affinity
   purification, mass spectrometry, metabolism, human tissues

Introduction

   UDP-glucuronosyltransferases (UGTs) are well known for their crucial
   role in the regulation of cellular homeostasis, by limiting stress
   induced by toxic drugs, other xenobiotics and endogenous lipophilic
   molecules, and by controlling the hormonal signaling network (Rowland
   et al., [33]2013; Guillemette et al., [34]2014). UGTs coordinate the
   transfer of the sugar moiety of their co-substrate UDP-glucuronic acid
   (UDP-GlcA) to amino, hydroxyl and thiol groups on a variety of
   lipophilic molecules, thereby reducing their bioactivity and
   facilitating their excretion. In humans, nine UGT1A and ten UGT2
   enzymes constitute the main glucuronidating enzymes. UGTs are found in
   nearly all tissues, each UGT displaying a distinct profile of tissue
   expression, and are most abundant in the liver, kidney and
   gastrointestinal tract, where drug metabolism is highly active. These
   membrane-bound enzymes localized in the endoplasmic reticulum (ER)
   share between 55 and 97% sequence identity, thus displaying substrate
   specificity and some overlapping substrate preferences (Rowland et al.,