Cytosolic PCNA interacts with p47phox and controls NADPH oxidase NOX2 activation in neutrophils

Abstract

Neutrophils produce high levels of reactive oxygen species (ROS) by NADPH oxidase that are crucial for host defense but can lead to tissue injury when produced in excess. We previously described that proliferating cell nuclear antigen (PCNA), a nuclear scaffolding protein pivotal in DNA synthesis, controls neutrophil survival through its cytosolic association with procaspases. We herein showed that PCNA associated with p47phox, a key subunit of NADPH oxidase, and that this association regulated ROS production. Surface plasmon resonance and crystallography techniques demonstrated that the interdomain-connecting loop of PCNA interacted directly with the phox homology (PX) domain of the p47phox. PCNA inhibition by competing peptides or by T2AA, a small-molecule PCNA inhibitor, decreased NADPH oxidase activation in vitro. Furthermore, T2AA provided a therapeutic benefit in mice during trinitro-benzene-sulfonic acid (TNBS)–induced colitis by decreasing oxidative stress, accelerating mucosal repair, and promoting the resolution of inflammation. Our data suggest that targeting PCNA in inflammatory neutrophils holds promise as a multifaceted antiinflammatory strategy.

PCNA has specific functions in regulation of metabolism in haematological cells

“…Several lines of evidence support cytosolic or non-canonical roles for PCNA: I) Cytosolic PCNA is reported to regulate apoptosis by binding to procaspases in mature neutrophils (Witko-Sarsat et al, 2010), multiple myeloma (MM) cells (Müller et al, 2013), and neuroblastoma cells (Yin et al, 2015); II) Inhibition of the ability of APIM-containing proteins to interact with PCNA by treating cells with a cellpenetrating peptide containing the APIM sequence (APIM-peptide) strongly reduced the secretion of cytokines from human monocytes stimulated with Toll-like receptor ligands (Olaisen et al, 2015); III) Cytosolic PCNA in cancer cells inhibits activation of natural killer cells, thereby helping cancer cells to evade the immune system (Rosental et al, 2011); IV) Glycolytic enzymes associate with PCNA, and nuclear export of PCNA correlated with increased Warburg-effect in acute myeloid leukaemia (AML) cells (Naryzhny & Lee, 2010;Ohayon et al, 2016); V) Multiple proteins involved in PI3K/AKT and MAPK signaling were found in PCNA pull-downs (Olaisen et al, 2015), and targeting PCNA has been linked to downregulation of EGFR and PI3K/AKT pathways in bladder cancer cells and monocytes (Olaisen et al, 2015;Sogaard, Blindheim, et al, 2018); VI) Cytosolic PCNA participates in reactive oxygen species (ROS) regulation via interaction with a subunit of the NADPH oxidase complex, p47phox (Ohayon et al, 2019); VII) Combination treatments with APIM-peptide and receptor tyrosine kinase inhibitors against EGFR/HER2/VEGFR and cMET showed increased anticancer activity in absence of any DNA-damaging agent (Sogaard et al, 2019); and VIII) A functional conservation of PCNA as a scaffold protein in MAPK signaling has been shown between mammalians and yeast (Olaisen et al, 2018). These cytosolic scaffold functions of PCNA, as well as the verified APIM-PCNA interactions important in DNA damage repair and/or tolerance, are illustrated in Figure 1.…”

“…(Bacquin et al, 2013;Fattah et al, 2014;Gilljam et al, 2009;Gilljam, Müller, Liabakk, & Otterlei, 2012;Raeder et al, 2018;Seelinger & Otterlei, 2020). Cytosolic roles: Multiple cytosolic proteins involved in cellular signaling and metabolism, including glycolytic enzymes, contain APIM or PIP-box motifs (Ohayon et al, 2019;Olaisen et al, 2018;Olaisen et al, 2015), or are found in complex with PCNA (Naryzhny & Lee, 2010;Olaisen et al, 2015;Witko-Sarsat et al, 2010). Studies have shown that impairing PCNA´s scaffold function affects multiple signaling pathways including the PI3K/AKT and MAPK pathways and the cytokine production after toll receptor stimuli (Olaisen et al, 2018;Olaisen et al, 2015;Sogaard et al, 2019), regulation of apoptosis (Müller et al, 2013;Witko-Sarsat et al, 2010;Yin et al, 2015), cytotoxicity of NK cells (Rosental et al, 2011;Shemesh et al, 2018), and cellular defense against ROS (Ohayon et al, 2019).…”

Network pharmacology analysis of the therapeutic mechanisms of the traditional Chinese herbal formula Lian Hua Qing Wen in Corona virus disease 2019 (COVID-19), gives fundamental support to the clinical use of LHQW

“…PCNA, a nuclear scaffolding protein pivotal in DNA synthesis, controls neutrophil survival through its cytosolic association with procaspases. Targeting PCNA in inflammatory neutrophils holded promise as a multifaceted anti-inflammatory strategy ( Ohayon et al., 2019 ). Oleanolic acid could downregulate the expression of PCNA to inhibit prostate cell proliferation ( Cheon et al., 2020 ).…”

Modulation of the Apurinic/Apyrimidinic Endonuclease Activity of Human APE1 and of Its Natural Polymorphic Variants by Base Excision Repair Proteins

“…Using the GRAMM-X web server, the WT APE1 structure (Protein Data Bank [PDB] ID 1DE8, [ 61 ]) was docked with OGG1 (PDB ID 1EBM, [ 62 ]), AAG (PDB ID 1F4R, [ 63 ]), UNG2 (PDB ID 1EMH, [ 64 ]), Polβ (PDB ID 4PHD, [ 65 ]), or PCNA (PDB ID 6FCM, [ 66 ]). Protein–protein docking of APE1 and XRCC1 was not performed because of the absence of a full-length XRCC1 structure.…”