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

Area of Science:

• Biochemistry
• Molecular Biology
• Cell Biology

Background:

• Pirin is an iron-binding protein with proposed roles in NFκB activation and quercetinase activity.
• Previous studies suggested pirin acts as a co-activator for p65 NFκB transcription factor.

Purpose of the Study:

• To investigate the interaction between pirin and Fe(III)-p65.
• To determine pirin's role in TNFα-activated gene transcription.
• To elucidate pirin's subcellular localization and confirm its quercetinase activity.

Main Methods:

• Analytical size exclusion chromatography (SEC) and fluorescence polarization (FP) assays.
• Analysis of TNFα-activated gene transcription in pirin knockout and knockdown cells.
• Immunofluorescence microscopy and cell fractionation.
• Biochemical assays for quercetinase activity and inhibitor studies.

Main Results:

• No interaction was detected between pirin and Fe(III)-p65 using SEC and FP.
• Pirin loss did not affect TNFα-activated p65-regulated gene transcription.
• Pirin predominantly localizes to the cytoplasm, specifically the endoplasmic reticulum (ER), not the nucleus.
• Pirin's quercetinase activity was confirmed, and its inhibition affected cellular quercetin levels.

Conclusions:

• The proposed mechanism of pirin as a p65 NFκB co-activator is not supported by these findings.
• Pirin's confirmed quercetinase activity and cytoplasmic localization suggest roles in small molecule binding and potentially ER-related functions.
• These results challenge the dominant model of pirin function and highlight its enzymatic activity.