Abstract:

My Ph.D. research focused on the study of the cytosolic Proliferating Cell Nuclear Antigen (PCNA) protein, whose function is to coordinate various effector mechanisms of neutrophils by associating with different partner proteins, particularly in the context of COVID-19 following infection by SARS- CoV-2.

The first part of my thesis, which concerns the study of neutrophils from COVID-19 patients, demonstrated that cytosolic PCNA is increased in the neutrophils of COVID-19 patients, depending on the severity of the disease. The amount of PCNA correlates with NADPH oxidase activity, and PCNA is associated with calprotectin, a dimeric protein composed of S100A8 and S100A9, which has been described as a predictive marker of mortality in patients.

Proteomic analysis of the neutrophil cytosols from COVID-19 patients revealed a strong regulation of interferon-induced proteins, and the interactome analysis of PCNA showed that PCNA was associated with some of these proteins (MX1, MX2, OAS3, and STAT1). Additionally, PCNA was associated with protein-arginine deiminase type 4 (PADI4) and Histone 3, which are involved in the formation of neutrophil extracellular traps (NET). In the case of Histone 3, its association with PCNA was only observed in the cytosols of neutrophils from patients in intensive care. These findings suggest that PCNA is involved both in the immune response and in the formation of pro-inflammatory mediators such as NET.

The second part of my thesis was initiated by the observation of an increased interaction between PCNA and NAMPT in the neutrophils of COVID-19 patients. Since NAMPT activity is associated with sirtuin 1, a NAD+-dependent lysine deacetylase, we investigated whether acetylation regulates ROS production by NADPH oxidase and if this regulation is dependent on the PCNA scaffold in neutrophils from healthy donors.

Our results first demonstrated that there is an interaction between PCNA and sirtuin 1 in the cytosol. When sirtuin-1 is inhibited by sirtinol, a dose-dependent decrease in ROS production via NADPH oxidase activity is observed. This inhibition does not affect MAP kinase signaling. Furthermore, co- immunoprecipitation with an anti-acetyl antibody identified targets of sirtuin 1, particularly coronin- 1A and S100A8, two modulatory proteins of NADPH oxidase, with S100A8 also being a partner of PCNA. We proposed a model in which the PCNA-sirtuin 1 association regulates the acetylation of PCNA partner proteins involved in NADPH oxidase, and this inhibitory regulation, which may be disrupted in the neutrophils of COVID-19 patients, could represent a new key mechanism in the regulation of PCNA- dependent neutrophil functions.