Background: The clonoSEQ® Assay (Adaptive Biotechnologies Corporation, Seattle, USA) identifies and tracks unique disease-associated immunoglobulin (Ig) sequences by next-generation sequencing of IgH, IgK, and IgL rearrangements and IgH-BCL1/2 translocations in malignant B cells. Here, we describe studies to validate the analytical performance of the assay using patient samples and cell lines.
Previous experiments performed in recombinant systems have suggested that protein–protein interactions occur between the UGTs and may play a significant role in modulating enzyme activity. However, evidence of UGT protein–protein interactions either in vivo or in more physiologically relevant in vitro systems has yet to be demonstrated. In this study, we examined oligomerization and its ability to affect glucuronidation in plated human hepatocytes. siRNA down regulation experiments and activity studies were used to examine changes in metabolite formation of one UGT isoform due to down regulation of a second UGT isoform. Selective siRNA directed towards UGT1A9 or UGT2B7 resulted in significant and selective decreases in their respective mRNA levels. As expected, the metabolism of the UGT1A9 substrate propofol decreased with UGT1A9 down regulation. Interestingly, UGT1A9 activity, but not UGT1A9 mRNA expression, was also diminished when UGT2B7 expression was selectively inhibited, implying potential interactions between the two isoforms. Minor changes to UGT1A4, UGT2B4 and UGT2B7 activity were also observed when UGT1A9 expression was selectively down regulated. To our knowledge, this represents the first piece of evidence that UGT protein–protein interactions occur in human hepatocytes and suggests that expression levels of UGT2B7 may directly impact the glucuronidation activity of selective UGT1A9 substrates.
Previous experiments performed in recombinant systems that over‐express either a single or multiple UDP‐glucuronosyltranseferase (UGT) isoforms have suggested that protein‐protein interactions occur between the UGTs and may play a significant role in modulating enzyme activity. However, evidence of UGT protein‐protein interactions either in vivo or in more physiologically relevant in vitro systems has yet to be demonstrated. In the current study, we examined oligomerization and its ability to affect glucuronidation in plated human hepatocytes. siRNA down regulation experiments and activity studies were used to examine changes in metabolite formation due to disrupted protein interactions. Selective siRNA down regulation of UGT1A9 or UGT2B7 resulted in significant and selective decreases in their respective mRNA levels. As expected, the metabolism of the UGT1A9 probe substrate propofol decreased with UGT1A9 down regulation. Interestingly, UGT1A9 activity was also diminished when UGT2B7 expression was selectively inhibited, implying potential interactions between the two isoforms. To our knowledge, this represents the first piece of evidence that UGT protein‐protein interactions occur in human hepatocytes and suggests that expression levels of UGT2B7 may directly impact the glucuronidation activity of selective UGT1A9 substrates such as propofol.
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