Pirin does not bind to p65 or regulate NFkappaB-dependent gene expression

Abstract

Pirin is a non-heme iron binding protein with a variety of proposed functions including serving as a co-activator of p65 NF{kappa}B and quercetinase activity. We report here, failure to confirm pirins primary proposed mechanism, binding of Fe(III)-pirin and p65. Analytical size exclusion chromatography (SEC) and fluorescence polarization (FP) studies did not detect an interaction. We also found no effects of pirin on TNF-activated p65-regulated gene transcription using mouse embryonic fibroblasts (MEFs) from a pirin knockout mouse and a pirin knockdown NIH3T3 fibroblast cell line. TNF - activated p65 response gene mRNA was neither increased nor decreased in cells with loss of pirin compared to wildtype 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 (PDI) as well as cyotoplasmic labeling. We confirmed pirins 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. Since pirin is localized to 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 pirins function (NF{kappa}B regulation) but confirm its quercetinase activity with implications for the mechanisms of pirin binding small molecules. Significance StatementPirin 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 pirins functional mechanism remains poorly understood. We raise doubts about the primary description of pirin as a nuclear regulator of p65 NF{kappa}B function but validate pirins role as a quercetinase. We show that pirin-binding compounds can raise cellular quercetin levels. Further studies will be required to fully understand pirins biological mechanisms.