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

Area of Science:

• Molecular biology
• Biochemistry
• Cell biology

Background:

• Pirin is an iron-binding protein with proposed roles in NFκB signaling and as a quercetinase.
• Its precise biological function and mechanism of action remain poorly understood, particularly regarding its interaction with p65 NFκB.
• Small molecule inhibitors targeting pirin have been identified, but their cellular effects are not fully elucidated.

Purpose of the Study:

• To investigate the proposed function of pirin as a coactivator of p65 NFκB.
• To confirm and characterize pirin's quercetinase activity and its implications for cellular quercetin levels.
• To determine pirin's subcellular localization and its potential role in ER stress.

Main Methods:

• Analytical size exclusion chromatography and fluorescence polarization to assess protein-protein interactions.
• Gene transcription assays using pirin knockout and knockdown cell lines.
• Immunofluorescence microscopy and cell fractionation to determine pirin localization.
• Biochemical assays to confirm quercetinase activity and inhibition.
• Analysis of ER stress response genes.

Main Results:

• No evidence of pirin binding to Fe(III) or interaction with p65 NFκB was detected.
• Pirin knockdown or knockout did not affect tumor necrosis factor α-induced p65-regulated gene transcription.
• Pirin predominantly localized to the cytoplasm, co-localizing with the endoplasmic reticulum, contrary to previous reports of nuclear localization.
• Pirin's quercetinase activity was confirmed, and inhibition of pirin increased cellular quercetin levels.
• Pirin knockdown did not impact ER stress signaling pathways.

Conclusions:

• The proposed function of pirin as a nuclear regulator of p65 NFκB is not supported by these findings.
• Pirin's role as a quercetinase is validated, with implications for modulating cellular quercetin levels.
• Pirin's cytoplasmic localization, particularly in the ER, suggests alternative biological functions.
• Pirin-binding compounds may represent a strategy to enhance cellular quercetin, relevant for cancer and inflammatory diseases.