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Alternative splicing at the human glucuronosyltransferase 1 gene locus (UGT1) produces alternate isoforms UGT1A_i2s that control glucuronidation activity through protein-protein interactions. Here, we hypothesized that UGT1A_i2s function as a complex protein network connecting other metabolic pathways with an influence on cancer cell metabolism. This is based on a pathway enrichment analysis of proteomic data that identified several high-confidence candidate interaction proteins of UGT1A_i2 proteins in human tissues-namely, the rate-limiting enzyme of glycolysis pyruvate kinase (PKM), which plays a critical role in cancer cell metabolism and tumor growth. The partnership of UGT1A_i2 and PKM2 was confirmed by coimmunoprecipitation in the HT115 colon cancer cells and was supported by a partial colocalization of these two proteins. In support of a functional role for this partnership, depletion of UGT1A_i2 proteins in HT115 cells enforced the Warburg effect, with a higher glycolytic rate at the expense of mitochondrial respiration, and led to lactate accumulation. Untargeted metabolomics further revealed a significantly altered cellular content of 58 metabolites, including many intermediates derived from the glycolysis and tricarboxylic acid cycle pathways. These metabolic changes were associated with a greater migration potential. The potential relevance of our observations is supported by the down-regulation of UGT1A_i2 mRNA in colon tumors compared with normal tissues. Alternate UGT1A variants may thus be part of the expanding compendium of metabolic pathways involved in cancer biology directly contributing to the oncogenic phenotype of colon cancer cells. Findings uncover new aspects of UGT functions diverging from their transferase activity., (Copyright © 2017 by The American Society for Pharmacology and Experimental Therapeutics.)

Transcripts of the UGT1A gene, encoding half of human UDP-glucuronosyltransferase (UGT) enzymes, undergo alternative splicing, resulting in active enzymes named isoforms 1 (i1s) and novel truncated isoforms 2 (i2s). Here, we investigated the effects of depleting endogenous i2 on drug response and attempted to unveil any additional biologic role(s) for the truncated novel UGT proteins. We used an integrated systems biology approach that combines RNA interference with unbiased global genomic and proteomic screens, and used HT115 colorectal cancer cells as a model. Consistent with previous evidence suggesting that i2s negatively regulate i1s through protein-protein interactions, i2-depleted cells were less sensitive to drug-induced cell death (IC50 of 0.45 ± 0.05 µM versus 0.22 ± 0.03 µM; P = 0.006), demonstrating that modulation of i2 levels meaningfully impacts drug bioavailability and cellular response. We also observed reduced production of reactive oxygen species by 30% (P < 0.05), and an enhanced expression (>1.2-fold; P < 0.05) of several proteins, such as hemoglobin α genes and superoxide dismutase 1, that have network functions associated with antioxidant properties. Interaction proteomics analysis of endogenous proteins from the cellular model, mainly in human intestine but also in kidney tissues, further uncovered interactions between i2s (but not i1s) and the antioxidant enzymes catalase and peroxiredoxin 1, which may influence antioxidant potential through sequestration of these novel partners. Our findings demonstrate for the first time dual roles for i2s in the cellular defense system as endogenous regulators of drug response as well as in oxidative stress.

Alternative splicing (AS) is one of the most significant components of the functional complexity of human UDP-glucuronosyltransferase enzymes (UGTs), particularly for the UGT1A gene, which represents one of the best examples of a drug-metabolizing gene regulated by AS. Shorter UGT1A isoforms [isoform 2 (i2)] are deficient in glucuronic acid transferase activity but function as negative regulators of enzyme activity through protein-protein interaction. Their abundance, relative to active UGT1A enzymes, is expected to be a determinant of the global transferase activity of cells and tissues. Here we tested whether i2-mediated inhibition increases with greater abundance of the i2 protein relative to the isoform 1 (i1) enzyme, using the extrahepatic UGT1A7 as a model and a series of 23 human embryonic kidney 293 clonal cell lines expressing variable contents of i1 and i2 proteins. Upon normalization for i1, a significant reduction of 7-ethyl-10-hydroxycamptothecin glucuronide formation was observed for i1+i2 clones (mean of 53%) compared with the reference i1 cell line. In these clones, the i2 protein content varied greatly (38-263% relative to i1) and revealed two groups: 17 clones with i2 < i1 (60% ± 3%) and 6 clones with i2 ≥ i1 (153% ± 24%). The inhibition induced by i2 was more substantial for clones displaying i2 ≥ i1 (74.5%; P = 0.001) compared with those with i2 < i1 (45.5%). Coimmunoprecipitation supports a more substantial i1-i2 complex formation when i2 exceeds i1. We conclude that the relative abundance of regulatory i2 proteins has the potential to drastically alter the local drug metabolism in the cells, particularly when i2 surpasses the protein content of i1.

Objective: UGT2B7 is a key member of the UDP-glucuronosyltransferase (UGT) family that participates in glucuronidation of endogenous compounds and pharmaceuticals. Much evidence suggests a large interindividual variability of UGT2B7-mediated glucuronidation, which is still unexplained by polymorphisms. We hypothesized that alternative splicing may be responsible for the variability in the UGT2B7 function., Methods: We carried out a comprehensive scan for additional exons at this locus and discovered multiple alternative splicing events. We then determined transcript expression profiles across a large variety of human tissues and assessed some of these variants for their glucuronidation activity in human cells., Results: In-depth analysis of the UGT2B7 gene structure led to the discovery of six novel exons. Kidney and liver samples presented the greatest enrichment of tissue-specific splicing, with 21 new UGT2B7 transcripts isolated. Furthermore, transcription from the proximal promoter (exon 1), associated with the active UGT2B7 mRNA isoform 1 (UGT2B7&#95;v1), is most commonly observed in the gastrointestinal tract, whereas a distal promoter (exon 1a) induces the exclusion of the canonical exon 1 and is active in hormone-related tissues. We also showed that novel transcripts with alternative 3' ends could be translated into proteins lacking glucuronosyltransferase activity in human cells but acting as negative regulators on transferase activity when coexpressed with the active UGT2B7 protein., Conclusion: Our findings point toward a significant variability in structure, abundance, and tissue-specific UGT2B7 transcriptome, in addition to novel functions for UGT2B7-derived proteins, all of which may ensure the production of tissue-specific proteomes and functions.