Pirin does not bind to p65 or regulate NFκB-dependent gene expression but does modulate cellular quercetin levels.

Pirin is a nonheme iron-binding protein with a variety of proposed functions, including serving as a coactivator of p65 NFκB and quercetinase activity. We report here, failure to confirm pirin's primary proposed mechanism, binding of Fe(III)-pirin and p65. Analytical size exclusion chromatography and fluorescence polarization studies did not detect an interaction. We also found no effects of pirin on tumor necrosis factor α-activated p65-regulated gene transcription using mouse embryonic fibroblasts from a pirin knockout mouse and a pirin knockdown NIH3T3 fibroblast cell line. Tumor necrosis factor α-activated p65 response gene mRNA was neither increased nor decreased in cells with loss of pirin compared to wild-type (WT) cells. Furthermore, pirin immunofluorescence in NIH3T3 fibroblasts showed primarily a cytoplasmic localization, not nuclear, as in most previous studies. This was confirmed by cell fractionation analysis. Pirin did show colocalization with the endoplasmic reticulum (ER) marker protein disulfide isomerase as well as cyotoplasmic labeling. We confirmed pirin's quercetinase activity in biochemical assays and demonstrated competitive inhibition by the pirin inhibitor, CCG-257081. Cellular quercetin levels in cells exposed to quercetin in vitro were increased by knockdown of pirin or by treatment with pirin inhibitors. Because pirin is localized to the ER and flavanols are protective of ER stress, we investigated whether pirin knockdown altered ER stress signaling, but did not find any effect of pirin knockdown on ER stress response genes. Our results challenge the dominant model of pirin's function (NFκB regulation) but confirm its quercetinase activity with implications for the mechanisms of pirin binding small molecules. SIGNIFICANCE STATEMENT: Pirin has multiple proposed functions and plays an important role in cancer (melanoma, colon, and breast) and inflammatory diseases. Small molecule pirin-binding compounds have been identified, but pirin's functional mechanism remains poorly understood. This study raises doubts about the primary description of pirin as a nuclear regulator of p65 NFκB function but validates pirin's role as a quercetinase. This study shows that pirin-binding compounds can raise cellular quercetin levels. Further studies will be required to fully understand pirin's biological mechanisms.